Selection marker gene free recombinant strains: method for obtaining them and the use of these strains

ABSTRACT

The present invention discloses a selection marker free system which can be used to introduce genetic modifications in bacteria, yeasts and fungi. The system can be employed to introduce or delete desired genes or DNA fragments in the genome of the indicated host species without leaving any undesired DNA i.e. the selection marker used for selection of transformants or other DNA used for cloning. In this way strains have been developed containing only desired genes introduced at desired chromosomal sites. Similarly, desired DNA fragments have been deleted or replaced at desired sites.

This application is a continuation of application Ser. No. 08/279,980,filed 22 Jul. 1994, now abandoned.

TECHNICAL FIELD

The present invention discloses selection marker gene free recombinantstrains, a method for obtaining these strains and the use of thesestrains. Furthermore, the method of the present invention is used forperforming strain improvement.

BACKGROUND OF THE INVENTION

There is an increasing social concern about the use of recombinant DNAtechnology. One of the promising application areas of recombinant DNAtechnology is strain improvement. Starting from the early days offermentative production processes there has been a demand for theimprovement of the productivity of the strains used for production.

Classical strain improvement programs for industrially employedmicroorganisms are primarily based on random mutagenesis followed byselection. Mutagenesis methods have been described extensively; theyinclude the use of UV light, NTG or EMS as mutagens. These methods havebeen described extensively for example in "Biotechnology: acomprehensive treatise in 8 vol." Volume I, Microbial fundamentals,Chapter 5b, Verlag Chemie GmbH, Weinheim, Germany.

Selection methods are generally developed around a suitable assay andare of major importance in the discrimination between wild type andmutant strains.

It has turned out that these classical methods are limited in theirpotential for improvement. Generally speaking consecutive rounds ofstrain improvement yield diminishing increases in yield of desiredproducts. This is at least partially due to the random character of themutagenesis methods employed. Apart from desired mutations these methodsalso give rise to mutations which are undesirable and which maynegatively influence other characteristics of the strains.

In view of these drawbacks it can be understood that the use ofrecombinant DNA methods was hailed as a considerable improvement. Ingeneral, recombinant DNA methods used in strain improvement programs aimat the increased expression of desired gene products.

The gene products may be proteins that are of interest themselves, onthe other hand it is also possible that the encoded gene products serveas regulatory proteins in the synthesis of other products.

Strains can be improved by introducing multiple copies of desiredprotein encoding genes into specific host organisms. However, it is alsopossible to increase expression levels by introducing regulatory genes.

Genes are introduced using vectors that serve as vehicles forintroduction of the genes. Such vectors may be plasmids, cosmids orphages. The vector may be capable of expression of the genes in whichcase the vector generally is self-replicating. The vector may howeveralso only be capable of integration. Another characteristic of thevector is that, when the expression product cannot be selected easilybased on altered phenotypic properties, the vector is equipped with amarker that can easily be selected for.

Vectors have not been isolated from all known microorganisms eithersince no vector could be found in the organism or since availablevectors from other organisms could be used with little or nomodification. The same applies to selection marker genes.

Widespread use and the subsequent spreading of specific marker genes hasrecently become debatable. This is especially due to the finding thatthe use of antibiotics and antibiotic selection markers gives rise to anundesired spread of strains that have become antibiotic resistant. Thisnecessitates the continued development of novel ever more potentantibiotics.

It is therefore not surprising that there is a general tendency in largescale production to use recombinant microorganisms containing noantibiotic resistance genes or more generally as little as possible offoreign DNA.

Ideally the transformed microorganism would contain only the desiredgene(s), fragments thereof or modifications in the gene and as little aspossible or no further remnants of the DNA used for cloning.

SUMMARY OF THE INVENTION

The present invention discloses a selection marker gene that can easilybe deleted again from the recombinant host organism. The deletion of thesaid marker gene is based on dominant selection.

The marker is used in species so diverse as bacteria, filamentous fungiand yeasts.

The advantageous activity of the selection markers used herein is basedon the following two step principle:

a) the gene is integrated into the genome of the host organism andrecombinant cells are selected,

b) the transformed cell is grown on a substrate, which is converted bythe marker gene encoded activity to a product that is lethal to thecell.

Selected cells will be recombinant and will have deleted the selectionmarker gene.

In general terms the present invention discloses cells, that may beanimal or plant cells, and microorganisms that have a modification inthe genome characterized in that the alteration is introduced using theamdS gene or the cDNA derived therefrom.

An example of a selection marker gene that can be used in this way isthe acetamidase gene. Preferably, this gene is obtainable fromfilamentous fungi, more preferably from Aspergilli, most preferably fromAspergillus nidulans.

The invention further shows the introduction, deletion or modificationof desired heterologous or homologous genes or DNA elements in the hostorganisms of choice using the acetamidase (amdS) gene as a marker.Subsequently the amdS gene is deleted. Preferably, the amdS and thedesired genes are introduced site-specifically.

The invention discloses a vector containing:

a) a desired DNA fragment destined for introduction into the hostgenome,

b) optionally a DNA sequence that enables the vector to integrate(site-specifically) into the genome of the host strain,

c) a gene encoding an acetamidase (e.g. the amdS gene from A.nidulans)between DNA repeats.

The invention further discloses host organisms transformed with the saidvector.

The invention further discloses selection marker gene free recombinantmicroorganisms.

Specifically, the invention discloses organisms containingsite-specifically introduced genes without any further foreign DNA beingpresent. The method is therefore also suited for repeated modificationsof the host genome, e.g. the sequential introduction of multiple genecopies at predetermined loci.

The invention provides a method for obtaining selection marker gene freerecombinant strains comprising the following steps:

a) integration into the genome of the strain of a desired DNA fragmentand a selection marker,

b) selection of the recombinants,

c) deletion of the selection marker preferably using internalrecombination between selection marker flanking repeats,

d) counter-selection based on the absence of the selection marker.

Although this is the preferred method for obtaining selection markergene free recombinant strain, the invention also provides modificationsof this method, for example: The desired DNA fragment and the selectionmarker may be present on two different DNA molecules which areco-transformed. The selection marker does not necessarily integrate intothe genome of the strain but may be present on an episomal DNA moleculewhich can be cured.

The present invention further illustrates that this marker gene can bedeleted from the genome of the transformed organisms without leaving atrace i.e. DNA used for cloning.

The present invention discloses the use of the amdS gene fromAspergillus as a marker in bacteria and yeast.

The invention discloses also the use of the amdS gene for deleting adesired gene from the chromosome of a `host` organism. Such modificationtechniques may be applied to filamentous fungi, yeasts and bacteria. Inspecific embodiments the following strains are employed Aspergilli,Trichoderma, Penicillium, Bacilli, E.coli, Kluyveromyces andSaccharomyces.

The method of the present invention provides recombinant strains withgenomic modifications obtained by repeating the procedure with the sameor other vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

Abbreviations used in the figures

Restriction enzymes and restriction sites

A=APaI; Ba=BamHI; B=BglII; Bs=BssHII; E=EcoRI;

H=HindIII; K =KpnI; N=NdeI; N=NotI; Ps=PstI;

P=PvuII; Sa=SalI; Sc=ScaI; S=SmaI; Sn=SnaBI;

Spe=SpeI; Sp=SphI; Ss=SstII; Xb=XbaI; X=XhoI.

Other

T.=LAC4 terminator sequence

FIG. 1: shows the restriction map of plasmid pamdS-1. This plasmidcontains the cDNA of the amdS gene from A.nidulans.

FIG. 2: shows schematically the marker gene free deletion of the glaAlocus from A.niger using the gene replacement vector pGBDEL4L. Theessential part of the gene replacement vector pGBDEL4L contains the amdSgene under control of the gpdA promoter cloned between repeats(3'-non-coding region of the glaA gene).

FIGS. 3a-3b, 4, 5, 6, 7a-7b, 8a-8b and 9: show schematically theconstruction pathway of pGBDEL4L as further outlined in Example 1 (FIG.6 is SEQ ID NO:3).

FIGS. 10a-10b

A. KpnI digests of pGBDEL4L transformants #41 (lane 1), #24 (lane 2),#23 (lane 3) and #19 (lane 4) and the host strain A.niger CBS 513.88(lane 5) and BamHI digests of pGBDEL4L transformants #41 (lane 6), #24(lane 7), #23 (lane 8), #19 (lane 9) and the host strain A.niger CBS513.88 (lane 10), probed with ³² P-labelled glaA promoter fragment andxylanase probe.

B. KpnI digests of GBA-102 (lane 1) and the GBA-102 strains afterfluoracetamide selection: GBA-107 (lane 2) and GBA-108 (lane 3) andBamHI digests of GBA-102 (lane 4) and the GBA-102 strains afterfluoracetamide selection: GBA-107 (lane 5) and GBA-108 (lane 6), probedwith ³² P-labelled glaA promoter fragment and xylanase probe.

FIG. 11:

A. Schematic presentation of BamHI and KpnI fragment lengths of thewild-type glaA locus in Aspergillus niger CBS 513.88.

B. Schematic presentation of BamHI and KpnI fragment lengths of thetruncated glaA locus in transformant #19 (=GBA-102).

C. Schematic presentation of BamHI and KpnI fragment lengths of thetruncated glaA locus in GBA-102 transformants after removal of the amdSgene (=GBA-107 and GBA-108).

FIGS. 12a-12b:

A: shows schematically the integration of the glaA gene into the 3'non-coding region of truncated glaA locus of A.niger GBA-107.

B: shows the result of the internal recombination between the 3' glaArepeats, flanking the amdS gene.

FIGS. 13, 14, 15, 16, 17a-17b, 18, 19, 20, 21, 22, 23a-23b and 24: showschematically the construction pathway of the integration vectorpGBGLA30 as further outlined in Example 2 (FIG. 14 is SEQ ID NO:10 andSEQ ID NO:11; FIG. 16 is SEQ ID NO:12 and SEQ ID NO:13; and FIG. 21 isSEQ ID NO:18 and SEQ ID NO:19).

FIG. 25: BglII digests of pGBGLA30 transformants #107-9 (lane 1), #107-7(lane 2) and #107-5 (lane 3), the host strain A.niger GBA-107 (lane 4)and the parental strain A.niger CBS 513.88 (lane 5) and KpnI digests ofpGBGLA30 transformants #107-9 (lane 6), #107-7 (lane 7) and #107-5 (lane8), the host strain A.niger GBA-107 (lane 9) and the parental strainA.niger CBS 513.88 (lane 10), probed with ³² P-labelled glaA promoterfragment.

FIG. 26a-26D

A: Schematic presentation of the KpnI and BglII fragment lengths of thewild-type glaA locus in Aspergillus niger CBS 513.88.

B: Schematic presentation of the KpnI and BglII fragment lengths of thetruncated glaA locus in Aspergillus niger GBA-107.

C: Schematic presentation of the KpnI and BglII fragment lengths of thetruncated glaA locus with a single copy pGBGLA30 integrated into theglaA 3'-non-coding region as in transformants #107-5 (=GBA-119) and#107-9 (=GBA-122).

D: Schematic presentation of the KpnI and BglII fragment lengths of thetruncated glaA locus in GBA-119 and GBA-122 transformants after removalof the amdS gene (=GBA-120, GBA-121, GBA-121 and GBA-124).

FIG. 27:

A: BglII digests of A.niger CBS 513.88 (lane 10), GBA-107 (lane 9),GBA-119 (lane 8) and the GBA-119 strains after fluoracetamide selection:#AG5-7 (=GBA-120) (lane 5), #AG5-5 (=GBA-121) (lane 6) and #AG5-6 (lane7); GBA-122 (lane 4) and the GBA-122 strains after fluoracetamideselection: #AG9-1 (=GBA-121) (lane 3), #AG9-2 (lane 2) and #AG9-4(=GBA-124) (lane 1), probed with ³² P-labelled 3" glaA non-codingfragment.

B: KpnI digests of A.niger CBS 513.88 (lane 10), GBA-107 (lane 9),GBA-119 (lane 8) and the GBA-119 strains after fluoracetamide selection:#AG5-7 (=GBA-120) (lane 5), #AG5-5 (=GBA-121) (lane 6) and #AG5-6 (lane7); GBA-122 (lane 4) and the GBA-122 strains after fluoracetamideselection: #AG9-1 (=GBA-121) (lane 3), #AG9-2 (lane 2) and #AG9-4(=GBA-124) (lane 1), probed with ³² P-labelled 3" glaA non-codingfragment.

FIG. 28: shows schematically the construction pathway of pGBGLA50.

FIGS. 29, 30a-30b, 31, 32a-32b and 33 show schematically theconstruction pathway of pGBGLA53.

FIG. 34: shows schematically the construction of pGBamdS1.

FIG. 35: shows schematically the construction of pGBamdS2.

FIG. 36: shows schematically the construction of pGBamdS3.

FIG. 37: shows schematically the construction of pGBamdS5.

FIG. 38: shows schematically the construction of pGBamdS6.

FIG. 39: shows schematically the construction of pPTLAC4 which was usedin the construction of pGBamdS6.

FIG. 40: shows schematically the construction of pGBamdS7.

FIG. 41: HindIII digests of K.lactis CBS 683 (lane 1), K.lactis CBS 2360(lane 2), the K.lactis CBS 683/pGBamdS1 transformants KAM-1 (lane 3),the K.lactis CBS 2360/pGBamdS1 transformant KAM-2 (lane 4) and the KAM-1strains after fluoracetamide selection (lane 5,6) probed with a ³² Plabelled LAC4 promoter fragment.

FIG. 42:

A: BamHI digests of K.lactis CBS 683/pGBamdS3 transformants (lanes 1-3)probed with a ³² P-labelled LAC4 terminator fragment.

B: BamHI digests of K.lactis CBS 683/pGBamdS5 transformants (lanes 1-5)and the host strain K.lactis CBS 683 (lane 6) probed with a ³²P-labelled LAC4 terminator fragment.

FIG. 43: BamHI digests of S.cerevisiae D273-10B (lane 1) andS.cerevisiae D273-10B/pGBamdS5 transformants (lanes 2-8) probed with a³² P-labelled amdS fragment.

FIG. 44: HindIII digests of K.lactis CBS 2360 (lane 1), the K.lactis CBS2360/pGBamdS6 transformant (lane 6) and strains from the K.lactis CBS2360/pGBamdS6 transformant after fluoracetamide selection (lanes 2-5)probed with a ³² P-labelled LAC4 terminator fragment.

FIG. 45: restriction map of the Bacillus plasmid pBHA1.

FIG. 46: restriction map of the Bacillus plasmid pLNF.

FIG. 47: shows schematically the construction of pGBamdS21.

FIG. 48: shows schematically the construction of pGBamdS22.

FIG. 49: shows schematically the construction of pGBamdS23.

FIG. 50: shows schematically the construction of pGBamdS25.

FIG. 51: shows schematically the construction of pGBamdS41.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses the use of a marker for selectingtransformed host strains. The selection marker gene can be used on anepisomal DNA vector. However, in the present invention, the marker geneis preferably integrated into the genome of the host strain. Theadvantage of the selection marker of the present invention is that it isa non-antibiotic dominant selection marker. Another advantage of theselection marker of the present invention is that it can be easilydeleted from the transformed host organism. The deletion of the markeris based on dominant selection. As such the selection marker of thepresent invention is a dominant and bi-directional selection marker. Toour knowledge it is the only selection-marker available which isbidirectional and dominant in both directions.

In the present description we use the term `selection marker gene`. Withthis term we mean the DNA coding for the marker protein in a functionalform irrespective of whether it is the actual gene or the cDNA derivedtherefrom. The gene or cDNA is used dependent on the host organism andthe expected splicing problems.

In the present invention we use the term `vector`. By this is intendedany DNA molecule that can be introduced into a selected hostirrespective of whether the vector integrates into the genome of thehost cell or remains episomal. The vector contains a selectable markergene functional in the selected host or can be co-transformed withanother DNA molecule containing such a selection marker gene.

The present description uses the term `desired heterologous orhomologous genes or DNA fragments`. By this is intended a DNA fragmentthat may be obtained from the host strain or from another species orstrain. The desired DNA fragment may contain any genetic element, partsthereof or combinations thereof, such as a gene (coding part or completelocus), a cDNA, a promoter, a terminator, an intron, a signal sequence,any regulatory DNA sequence or recognition sequence of DNA-bindingproteins. The fragment may also be a DNA sequence that has been modifiedi.e. contains one or more nucleotide alterations (e.g. insertions,deletions, substitutions).

The present description further uses the term `introduction` of adesired gene or DNA fragment. By this is intended an insertion,deletion, substitution of desired DNA sequences in a selected host cell.

The term `genetic modification` used in the present invention refers toany modification of DNA sequences in a selected host cell which is theresult of the introduction of any one of the above mentioned desired DNAfragments into the host cell, preferably by transformation orco-transformation.

In general all these genetic modifications can be performed using themethod of the present invention with subsequent deletion of theselection marker gene. Due to the fact that the recombinant straincontaining such a genetic modification does not contain the selectionmarker gene, the procedure of the present invention can be repeated, sothat the modifications suggested above can be combined in therecombinant strain. Ultimately, the procedure of the present inventioncan be used repeatedly up to the point that a recombinant strain isobtained from which all undesired activities have been removed bydeletion or inactivation of the corresponding genetic elements and whichcontains the desired activities at the desired levels by sequentialintroduction of the corresponding desired DNA fragments at desiredcopynumbers and preferably at desired and defined loci.

The A. nidulans acetamidase (amdS) gene allows A. nidulans to grow onacetamide as the sole N-source. For microorganisms that lack thepossibility or only have a very limited capacity to use acetamide as thesole N-source the acetamidase gene can in principle be used as aselection marker provided that acetamide is taken up by the cells. TheamdS gene has successfully been employed as a marker gene in Aspergilli(Kelly and Hynes (1985) EMBO J. 4, 475-479; Christensen et al. (1988)Bio/technology 6, 1419-1422), Penicillium (Beri and Turner (1987) Curr.Genet. 11, 639-641) and Trichoderma (Pentilla et al. (1987) Gene 61,155-164).

The present invention for the first time discloses the use of the amdSgene from A.nidulans as a selection marker in organisms other thanfilamentous fungi. The use of this selection marker is disclosed inbacteria and yeasts. Specifically, the use is demonstrated inS.cerevisiae, in K.lactis in B.subtilis, in B.licheniformis and inE.coli. In view of the disclosed applicability of the selection markerin species selected from such diverse groups as fungi, yeasts andbacteria it is to be expected that the marker will also be applicable inother species pertaining to these groups. Use of this marker istherefore not restricted to the disclosed species.

The amdS gene from A.nidulans is capable of converting acetamide toammonia and acetic acid. This property enables A.nidulans to grow on amedium containing acetamide as the sole N-source or C-source.

Another property of the amdS gene is that it is also able to convertfluoracetamide to ammonia and fluoracetic acid. Fluoracetic acid howeveris toxic to the cell. It is this property that forms the basis foranother aspect of the present invention i.e the production of markergene free recombinant strains. The fluoracetamide converting propertyenables the counter-selection of transformed cells. The amdS gene isintroduced into the host strain and integrated into the genome throughhomologous recombination. The transformed strains are selected on amedium containing acetamide as the sole N-source. Subsequently theselected strains are grown on a medium containing fluoracetamide andurea (or other preferably defined N-sources) as the sole N-sources. Thesurviving strains will have deleted the amdS gene.

The present invention uses the A.nidulans amdS gene as acetamidasemarker gene. The relevant properties provided by the acetamidase encodedby the A.nidulans amdS gene, i.e. the ability to hydrolyse acetamideinto ammonia and acetate as well as the ability to liberate fluoraceticacid from fluoracetamide, can also be provided by acetamidases fromother sources. Use of an acetamidase marker gene is therefore notrestricted to the A.nidulans amdS gene but includes any DNA sequenceencoding a functional acetamidase.

The frequency of marker deletion is substantially increased byincreasing the capacity of the gene for intrachromosomal homologousrecombination. To achieve this the amdS gene is preferably placedbetween DNA repeats. These repeats are not necessarily both present inthe vector but may also be created by a single cross-over integration.Alternatively, one may omit flanking repeats and rely on othermechanisms for removal or inactivation of the marker gene. In that case,however, the outcome may be less predictable and may not result inremoval but rather in mere inactivation of the marker gene.

The vector may be constructed in such a way that, after deletion of themarker gene, no extraneous foreign DNA (except the DNA of interest)remains in she chromosome of the host strain. The invention discloses avector comprising:

a) a desired DNA fragment destined for introduction into the hostgenome,

b) optionally a DNA sequence that enables the vector to integrate(site-specifically) into the genome of the host strain,

c) a gene encoding an acetamidase (e.g. the amdS gene from A.nidulans)between DNA repeats.

Identical results may be obtained when the DNA-fragment destined forintroduction into the host genome and the selectable marker gene (e.g.the acetamidase gene) are present on two different DNA molecules whichare co-transformed, in which case the DNA molecule containing theselectable marker does not necessarily integrate into the host genomebut may be present on an episomal DNA molecule which can be cured.

The sequences used for integration as mentioned under b) are used ifsite-specific (or better locus specific) integration is desired. If sucha sequence is not present the vector nevertheless may integrate into thegenome. This does not influence the ability to delete the selectionmarker gene.

The dominant counter-selection described above can be employed in thedevelopment of industrial production strains in various ways. The use ofa dominant selection marker is especially advantageous in thedevelopment of improved production strains due to the fact that thesestrains are often diploid or polyploid.

The vector used for integration of the amdS gene preferably containsanother gene of interest. The invention thus further enables theintroduction of desired foreign or homologous genes or DNA elements inthe host organisms of choice using the amdS gene as a marker.Subsequently the amdS gene is deleted. Preferably, the amdS and thedesired genes or DNA elements are introduced site-specifically,whereafter the amdS gene is deleted.

Specifically, the invention discloses organisms containingsite-specifically introduced genes without any further foreign DNA beingpresent. The invention is used for integration of multiple copies of adesired gene or a DNA element at predetermined genomic loci.

The invention provides a method for obtaining selection of marker genefree recombinant strains comprising the following steps:

integration of a desired gene or DNA element and a selection marker byhomologous recombination between sequences incorporated in an expressioncassette and sequences on the host chromosome,

selection using the selection marker gene that is dominant,

deletion of the selection marker gene using selection marker geneflanking regions,

selection based on the absence of the selection marker gene(counter-selection).

The present invention further shows that this marker gene can be deletedfrom the chromosomes of the transformed organisms without leaving atrace i.e. DNA used for cloning. Moreover, the invention also shows thatsimilar if not identical results can be obtained when the desired geneor DNA element and the selection marker are present on two different DNAmolecules which are co-transformed.

Finally the invention discloses the use of the amdS gene for deleting adesired gene from the chromosome of a `host` organism.

In view of the above, the method of the present invention is ideallysuited for, but not limited to the cloning and expression of genescoding for proteins used in food, feed or pharmaceutical applications orgenes involved in biosynthesis of antibiotics and other bio-activecompounds, i.e. recombinant proteins and/or hosts-organisms that aresubject to strict registration requirements.

Examples of such proteins are well known in the art and includechymosin, phytase, xylanases, amylases, cellulases and hemicellulases,cytokines and other pharmaceutical proteins, etc.

The same method is employed for deletion of genes coding for proteinsthat influence production levels of desired proteins again withoutleaving a marker gene in the genome. Such proteins include proteaseswhich actively digest the desired products that are highly expressed inthe host strain and that therefore have a reduced potential of producingand or secreting the desired proteins. A preferred method for thedeletion of a given gene would use a DNA construct containing thefollowing elements in a 5' to 3' order: sequences 5' of the gene to bedeleted, directly fused to sequences 3' of the gene to be deleted,followed downstream by a functional selection marker gene (preferably anacetamidase gene), followed downstream by again sequences 3' of the geneto be deleted. In this case both sequences 3' of the gene to be deletedare chosen such that they form repeats flanking the selection markergene. Transformation of this DNA construct and subsequent replacement ofthe chromosomla copy of the gene to be deleted by the DNA construct withcross-over points in the sequences 5' and 3' of the gene to be deletedresults in deletion of the given gene. Subsequent intrachromosomalrecombination between the repeats flanking the selection marker gene andcounter-selection for these recombinants finally results in a selectionmarker free strain with the given gene deleted. The DNA construct usedfor this deletion can be constructed such that no foreign DNA or othertraces of the genetic modification are left in the strain carrying thedeletion.

The invention discloses selection marker gene free recombinantmicroorganisms. Such microorganisms can be organisms that, after the useof the disclosed technology, contain an extra copy of a desired gene(either homologous or heterologous). Such microorganisms can bere-transformed over and over by sequential application of the sametechnology to insert or delete additional copies of the same or othergene(s) of interest.

The microorganisms may also be characterized in that they have (a)predetermined gene(s) deleted or altered in any desired way.

The method of the present invention makes possible the fine-tuning ofthe production of desired proteins. This possibility is based on theease with which repeated rounds of insertion and deletion can beperformed. The method makes possible the insertion or deletion of adesired number of gene copies. Thus the proteins are produced in desiredamounts and in desired ratios. This is especially useful for theproduction of mixtures of proteins or enzymes.

Whereas it is known that the acetamidase gene is capable of conversionof acetamide as the sole N-source in Aspergillus it is here shown thatthe acetamidase gene is easily deleted from the genome of transformedAspergilli. To achieve this the amdS gene is cloned between directrepeats. In principle any direct repeat which allows for internalrecombination can be employed. In the present examples this isdemonstrated by cloning the amdS gene between 3' amyloglucosidase (glaA)non-coding DNA sequences.

It is shown that the amdS gene can be integrated and deleted uponplating on medium containing fluoracetamide and urea as N-sources.

It is further demonstrated that the amyloglucosidase gene can be deletedfrom the genome of Aspergillus. A replacement vector is constructedcontaining a part of the glaA promoter, a synthetic DNA sequencecontaining stop codons in all three reading frames, the amdS gene fromA. nidulans under the control of the A.nidulansglyceraldehyde-3-phosphate dehydrogenase promoter and wherein the amdSgene is flanked by 3' glaA non-coding sequences. After transformation ofA.niger the vector is integrated by double crossing-over therebyeffectively replacing the amyloglucosidase gene. After selection foramdS activity the transformed strains are plated on fluoracetamide andurea. Selection resulted in strains wherein the amdS gene was deleted.

This example is an illustration of the possibility of using the amdSgene for deletion of a desired gene from the genome of an Aspergillusstrain. Other genes can be eliminated or modified in a similar manner.

In a further example it is demonstrated that a gene can be insertedmarker free at a predetermined site in the genome. An integration vectoris constructed containing the A.niger glaA locus and the amdS geneflanked by two 3' glaA non-coding repeats.

The construct is shown to integrate at the amyloglucosidase locus. Afterselection on fluoracetamide the amdS gene is deleted. In this way a genecopy is integrated at a specific locus without leaving marker DNA.

It is evident from the above that the procedures described herein enableone of skill in the art to integrate or delete desired genes atpredetermined loci without leaving selection marker DNA behind.

This method can be employed for gene amplification and gene replacement.

An especially important application would be the integration of desiredgenes. Followed by classical strain improvement whereafter the genesthat may be adversely affected by the classical strain improvementtechniques are replaced with fresh unaffected copies of the gene ofinterest without loss of expression level.

The system as described for Aspergillus above is expected to give thesame results when other fungal strains are employed, which are known tobe incapable of growth on acetamide as the sole N-source. The use of theamdS gene as a selection marker has been described for among othersPenicillium and Trichoderma. Moreover, the amdS gene can even be used infilamentous fungi which are capable of using acetamide as sole N-sourcealbeit poorly. In this case the background of poorly growinguntransformed cells can be repressed by the inclusion of CsCl in theselection media (Tilburn, J. et al. (1983) Gene, 26, 205-221). Hence thesystem is expected to be applicable to filamentous fungi in general.

In one embodiment of the present application it is surprisinglydemonstrated that the A.nidulans amdS gene can be used as a selectionmarker in K.lactis. In this Example it is shown that two differentK.lactis strains cannot grow on acetamide as the sole N-source. The twoK.lactis strains are plated on YCB medium which is

a) complete but without N-source,

b) as a) but with acetamide,

c) as a) but with ammonium sulphate.

It is shown that the strains do not grow on the medium under b) but dogrow on medium under c). Hence provided that the acetamide is taken upby the yeast cells and that the amdS gene can be expressed in K.lactisthe system is applicable in yeasts also at least as a selection marker.Concerning the counter-selection using fluoracetamide some furtherrequirements have to be met. Fluoracetate is toxic when activated by theenzyme acetyl-CoA-synthetase. Prerequisites for the fluoracetamidecounter-selection to also work on amdS⁺ yeasts are therefore

1) fluoracetamide should not be toxic for amdS⁻ yeasts,

2) the yeast cell wall and plasmamembrane should be permeable tofluoracetamide and

3) the enzyme acetyl-CoA-synthetase should be active.

To test this the amdS gene was cloned in K. lactis.

To avoid any potential splicing problems of the A.nidulans amdS gene inK.lactis the amdS cDNA from A.nidulans was cloned as shown in theExperimental section.

Subsequently the amdS was cloned downstream of a yeast promoter (LAC4,ADH1, KlEF) in a vector containing another marker(phosphotransferase-G418). This cloning is described in Example 8. Thevectors containing both the G418 marker and the amdS gene were selectedusing the G418 marker and were then used to optimize selectionconditions for the amdS+ phenotype.

Direct selection of K.lactis is shown in another embodiment of thepresent invention and for S.cerevisiae direct selection is shown inExample 11.

Subsequently it is demonstrated that counter-selection can be employedon the transformed yeast strains to remove the amdS gene.

The amdS gene system is used for both marker gene free insertion andmarker gene free deletion of a gene in yeast.

In a further embodiment the lactase gene is deleted from K.lactiswhereas in Example 14 a copy of the chymosin gene is inserted into theK.lactis genome.

The genes used here for insertion and deletion are only used asexamples. The same technology can be applied using other genes or DNAelements. As mentioned before the DNA fragments used for insertion ordeletion can be mutated genes, promoter sequences, regulatory sequencesetc. In all cases it is possible to insert or delete these sequences atdesired genomic sites and in desired numbers, without leaving a markergene behind.

The feasibility of the use of this system in other yeast strains isevident.

As a first step for use of the system of the present invention inbacteria it is shown in Example 15 that Bacillus subtilis and E.colicannot grow on acetamide as the sole N-source.

Example 16 describes the vectors that have been constructed for use inBacillus and E.coli.

It is demonstrated in Examples 17 and 18 that the amdS gene can beeffectively used in Bacillus and E.coli as selection marker, whereasExample 19 demonstrate the fluoracetamide counter-selection of bacterialamdS⁺ transformants.

The advantages of the system of the present invention are manifold. Themost striking advantages are given below:

It is demonstrated that the amdS system is universally applicable (plantcells, animal cell, yeasts, bacteria and filamentous fungi etc.),requiring only that the host in question cannot or only poorly grow onacetamide as sole C- or N-source but can utilize either acetate orammonia as sole C- or N-source, respectively.

The amdS system represents the only bi-directional and dominantselection system. This feature is extremely convenient for use in poly-or aneuploid strains which often is the case with natural isolatesand/or industrial strains.

After classical strain improvement any mutated copies of the desiredgene can be easily replaced by unmutated copies by gene replacement dueto the fact that the desired genes have been integrated at well-definedloci. The genes are thus replaced with unmutated genes without affectingthe expression level.

Due to the ability to introduce multiple integrations at well-definedand therefore non-random loci one can be assured that no undesirabletraits arise in the strain upon gene amplification.

The growing concern about the release of various selection markers inthe environment is overcome by the presented system. No selection markergene or other unnecessary or undesired DNA sequences need to present inthe production strains after introduction of the desired genes or othergenetic modifications.

Experimental

General molecular cloning techniques

In the examples described herein, standard molecular cloning techniquessuch as isolation and purification of nucleic acids, electrophoresis ofnucleic acids, enzymatic modification, cleavage and/or amplification ofnucleic acids, transformation of E.coli, etc., were performed asdescribed in the literature (Sambrook et al. (1989) "Molecular Cloning:a laboratory manual", Cold Spring Harbour Laboratories, Cold SpringHarbour, N.Y.; Innis et al. (eds.) (1990) "PCR protocols, a guide tomethods and applications" Academic Press, San Diego). Synthesis ofoligo-deoxynucleotides and DNA sequence analysis were performed on anApplied Biosystems 380B DNA synthesizer and 373A DNA sequencer,respectively, according to the user manuals supplied by themanufacturer.

Transformation of A.niger

Transformation of A.niger was performed according to the methoddescribed by Tilburn, J. et.al. (1983) Gene 26, 205-221 and Kelly, J. &Hynes, M. (1985) EMBO J., 4, 475-479 with the following modifications:

spores were grown for 16 hours at 30° C. in a rotary shaker at 300 rpmin Aspergillus minimal medium.

Aspergillus minimal medium consists of the following components: Perliter: 6 g NaNO₃ ; 0.52 g KCl; 1.52 g KH₂ PO₄ ; 1.12 ml 4M KOH; 0.52 gMgSO₄.7H₂ O; 10 g glucose; 1 g casamino acids; 22 mg ZnSO₄.7H₂ O; 11 mgH₃ BO₃ ; 5 mg FeSO₄.7H₂ O; 1.7 mg CoCl₂.6H₂ O; 1.6 mg CuSO₄.5H₂ O; 5 mgMnCl₂.4H₂ O; 1.5 mg Na₂ MoO₄.2H₂ O; 50 mg EDTA; 2 mg riboflavin; 2 mgthiamine.HCl; 2 mg nicotinamide; 1 mg pyridoxine.HCl; 0.2 mg panthotenicacid; 4 μg biotin; 10 ml Penicillin (5000 IU/ml)/Streptomycin (5000UG/ml) solution (Gibco).

only Novozym 234 (Novo Industri), and no helicase, was used forformation of protoplasts;

after protoplast formation (60-90 minutes), KC buffer (0.8M kCl, 9.5 mMcitric acid, pH6.2) was added to a volume of 45 ml. and the protoplastsuspension was centrifuged at 2500 g at 4° C. for 10 minutes in aswinging-bucket rotor. The protoplasts were resuspended in 20 ml. KCbuffer. Then, 25 ml of STC buffer (1.2M sorbitol, 10 mM Tris-HCl pH7.5,50 mM CaCl₂) was added and subsequently the protoplast suspension wascentrifuged at 2500 g at 4° C. for 10 minutes in a swinging-bucketrotor, washed in STC-buffer and resuspended in STC-buffer at aconcentration of 10⁸ protoplasts/ml;

to 200 μl of the protoplast suspension the DNA fragment, in a volume of10 μl in TE buffer (10 mM Tris-HCl pH7.5, 0.1 mM EDTA), was added andsubsequently 100 μl of a PEG solution (20% PEG 4000 (Merck), 0.8Msorbitol, 10 mM Tris-HCl pH7.5, 50 mM CaCl₂);

after incubation of the DNA-protoplast suspension at room temperaturefor 10 minutes, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mMTris-HCl pH7.5, 50 mM CaCl₂) was added slowly, with repeated mixing ofthe tubes. After incubation at room temperature for 20 minutes, thesuspensions were diluted with 5 ml STC buffer, mixed by inversion andcentrifuged at 2000 g at room temperature for 10 minutes. Theprotoplasts were resuspended gently in 1 ml 1.2M sorbitol and platedonto selective regeneration medium consisting of Aspergillus minimalmedium without riboflavin, thiamine.HCl, nicotinamide, pyridoxine.HCl,panthotenic acid, biotin, casamino acids and glucose but with 10 mMacetamide as the sole nitrogen source, 1M sucrose, solidified with 2%bacteriological agar #1 (Oxoid, England). Following growth for 6-10 daysat 30° C., the plates were replica plated onto selective acetamideplates consisting of Aspergillus selective regeneration medium with 2%glucose instead of sucrose and 1.5% agarose instead of agar. Singletransformants were isolated after 5-10 days of growth at 30° C.

Transformation of A. oryzae

Transformation of A. oryzae was performed according to the methoddescribed by Christensen, T. et al. in European Patent Application 0 238023 A2.

Transformation of T. reesei

Transformation of T. reesei was performed according to the methoddescribed by Penttilla M., Knowles, J. (1987) Gene 61 155-164.

Transformation of P. chrysogenum

The Ca-PEG mediated protoplast transformation procedure is used.Preparation of protoplasts and transformation of P.chrysogenum wasperformed according to the method described by Gouka et al., Journal ofBiotechnology 20(1991), 189-200 with the following modifications:

After transformation, the protoplasts were plated onto selectiveregeneration medium plates consisting of Aspergillus minimal medium,osmotically stabilized with 1.2M sucrose, containing 0.1% acetamide assole nitrogen source and solidified with 1.5% bacteriological agar #1(Oxoid, England).

After 5-8 days of incubation at 25° C. transformants appeared.

Transformation of K.lactis

The yeast K.lactis was transformed using the lithium acetate proceduredescribed by Ito H. et al. (1983) J. Bacteriol. 153, 163-168 with thefollowing modifications:

For transformation a K.lactis culture was taken with an OD₆₁₀ between0.5 and 1.0.

After the 5 minutes heatshock of the transformed cell suspensions, 1 mlYEPD/YNB (1% yeast-extract, 2% Bacto-peptone, 2% glucose and 0.17% YeastNitrogen Base w/o amino acids (YNB; Difco) was added and thecell-suspensions were incubated at 30° C. in a shaker incubator for150-180 minutes.

After the above mentioned incubation (at 30° C. for 150-180 minutes),the cell-suspensions were centrifuged at 2000 g at room temperature for5 minutes and plated on YEPD/G418 double layer medium solidified with 2%Bacto-agar (Difco). YEPD/G418 double layer plates were prepared asfollowed: 10 minutes prior to plating of the cell-suspensions 15 ml YEPDagar (1% yeast-extract, 2% Bacto-peptone, 2% glucose solidified with 2%Bacto-agar (Difco)) without G418 was poured onto 15 ml YEPD agar, whichcontained 50 μg G418/ml. This results in YEPD/G418 double layer plateswhich contain 25 μg G418/ml after diffusion of the antibiotic. TheYEPD/G418 double layer plates contained 25 μg G418/ml or 100 μg G418/mlin case of strains K.lactis CBS 683 or CBS 2360, respectively.

Isolation of DNA from Aspergillus, Trichoderma, Penicillium and yeast

The isolation of DNA from Aspergillus and Trichoderma was performedaccording to the procedure as described by Yelton, et al. (1984), Proc.Natl. Acad. Sci. 81, 1470-1474.

The isolation of DNA from Penicillium was performed according to theprocedure described by Kolar et al., Gene 62 (1988), 127-134.

The isolation of DNA from K.lactis or S.cerevisiae was performedaccording to the procedures described by Fujimura and Sakuma (1993),Biotechniques 14, 538.

Bacillus transformation and DNA-isolation

Transformation of the different Bacillus species as well as isolation ofplasmid or chromosomal DNA from these species was performed as describedby Bron (1990) "Plasmids" In: Molecular Biological Methods for Bacillus,Harwood, CR and Cutting, SM, eds., series Modern MicrobiologicalMethods, John Wiley & Sons, Chichester, UK.

For the transformation of B.subtilis BS-154 (CBS 363.94) competent cellswere used and for the transformation of B.licheniformis T5 (CBS 470.83)protoplast transformation was used. In the case of neomycin selection aconcentration of 20 μg/ml was used. For acetamide selection ofB.subtilis transformants, minimal medium agar was used in which casaminoacids and yeast extract were replaced by 20 mM acetamide. For acetamideselection of B.licheniformis transformants, protoplast regenerationmedium was used in which ammonium sulphate was replaced by 20 mMacetamide.

Removal of the amdS selection marker

The amdS marker in most examples relating to Aspergillus, Trichodermaand Penicillium is cloned between repeats consisting of a part of the 3'non-coding region of amyloglucosidase gene. Removal of the amdSselection marker is achieved either by internal recombination betweenthe 3' glaA non-coding repeats that flank the amdS selection marker orby homologous recombination between the repeats that are created byintegration via a single cross-over event. Selection of cells that havelost the amdS selection marker is achieved by growth on platescontaining fluoracetamide. Cells harbouring the amdS gene metabolizefluoracetamide to ammonium and fluoracetate which is toxic to the cell.Consequently, only cells that have lost the amdS gene are able to growon plates containing fluoracetamide.

In case of removal of the amdS marker from Aspergillus transformants,spores from these transformants were plated onto selective regenerationmedium (described above) containing 32 mM fluoracetamide and 5 mM ureuminstead of 10 mM acetamide, 1.1% glucose instead of 1M sucrose and 1.1%instead of 2% bacteriological agar #1 (Oxoid, England). After 7-10 daysof growth at 35° C. single colonies were harvested and plated onto 0.4%potato dextrose agar (Oxoid, England). In case of removal of the amdSmarker from Trichoderma transformants, spores of these transformantswere plated onto non selective minimal medium plates (per liter: 20 g.glucose, 5 g. (NH₄)₂ SO₄, 15 g. KH₂ PO₄, 0.6 g. MgSO₄, 0.6 g. CaCl₂,0.005 g. FeSO₄.7H₂ O, 0.0016 g. MnSO₄.H₂ O, 0.0014 g. ZnSO₄.7H₂ O, 0.002g. CoCl₂ ; pH5.5) supplemented with 10 mM fluoracetamide. After 5-10days at 30° C., colonies were harvested and plated onto 0.4% potatodextrose agar (Oxoid, England).

In case of removal of the amdS marker from Penicillium transformants,spores from these transformants were plated on selective medium platesconsisting of Aspergillus minimal medium with 10 mM fluor-acetamide and5% glucose, solidified with 1.5% bacteriological agar #1 (Oxoid,England). After 5-10 days of growth at 25° C. resistant coloniesappeared.

Determination of glucoamylase production by A.niger transformants

Of recombinant and control A.niger strains spores were collected byplating spores or mycelia onto PDA-plates (Potato Dextrose Agar, Oxoid),prepared according to the supplier's instructions. After growth for 3-7days at 30° C. spores were collected after adding 0,01% Triton X-100 tothe plates. After washing with sterile water approximately 10⁷ spores ofselected transformants and control strains were inoculated into shakeflasks, containing 20 ml of liquid pre-culture medium containing perliter: 30 g maltose.H₂ O; 5 g yeast extract; 10 g hydrolysed casein; 1 gKH₂ PO₄ ; 0.5 g MgSO₄.7H₂ O; 3 g Tween 80; 10 ml Penicillin (5000IU/ml)/Streptomycin (5000 UG/ml); pH 5.5. These cultures were grown at34° C. for 20-24 hours. 5-10 ml of this culture was inoculated into 100ml of fermentation medium containing per liter: 70 g maltodextrines; 25g hydrolysed casein; 12.5 g yeast extract; 1 g KH₂ PO₄ ; 2 g K₂ SO₄ ;0.5 g MgSO₄.7H₂ O; 0.03 g ZnCl₂ ; 0.02 g CaCl₂ ; 0.01 g MnSO₄.4H₂ O; 0.3g FeSO₄.7H₂ O; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml);adjusted to pH 5.6 with 4N H₂ SO₄. These cultures were grown at 34° C.for 5-10 days. Samples were taken for the analysis of the glucoamylaseproduction at different time points during fermentation. Fermentationbroth samples were centrifuged (10 minutes, 10.000×g) and supernatantscollected.

The glucoamylase activity was determined by incubating 10 μl of a sixtimes diluted sample of the culture supernatant in 0.032M NaAC/HACpH4.05 with 115 μl of 0.2% (w/v) p-Nitrophenyl α-D-glucopyranoside(Sigma) in 0.032M NaAc/HAc pH 4.05. After a 30 min incubation at roomtemperature, 50 μl of 0.3M Na₂ CO₃ was added and the absorption at awavelength of 405 nm was measured. The A_(405nm) is a measure for the AGproduction.

Cloning of the amdS cDNA

The A.nidulans amdS gene contains three small introns (Corrick et al.(1987) Gene 53, 63-71). In order to avoid problems caused by incorrectsplicing of these introns in yeast or lack of splicing in bacteria, wehave used an amdS cDNA for expression in yeasts and bacteria. Cloning ofthe amdS cDNA from an A.nidulans polyA⁺ RNA preparation has beendescribed by Corrick et al. ((1987), Gene 53, 63-71). In this example wehave used the A.niger NRRL 3135 transformant #4, which is transformed bymultiple copies of the A.nidulans amdS gene containing plasmid pAF2-2S(van Hartingsveld et al (1993). Gene 127, 87-94). Total RNA was isolatedby a direct LiCl precipitation according to a procedure modified fromAuffray et al. ((1980) Eur.J.Biochem. 107, 303-314). A.niger spores wereallowed to germinate and were grown overnight at 37° C. in a minimalmedium (Cove (1966) Biochim. Biophys. Acta 113, 51-56) supplemented withglucose as carbon source and with acetamide as sole nitrogen source.Mycelium was obtained and dried by filtration and subsequently frozenwith liquid nitrogen to be grounded. The powder was dispersed in 3MLiCL, 6M urea at 0° C. and maintained overnight at 4° C. Total cellularRNA was obtained after centrifugation at 16.000 g for 30 minutes and twosuccessive extractions with phenol/chloroform/isoamylalcohol (50:48:2).The RNA was precipitated with ethanol and dissolved in 1 ml 10 mMTris-HCL (pH7.4), 0.5% SDS. For polyA⁺ selection the total RNA samplewas heated for 5 minutes at 65° C. and subsequently applied to anoligo(dT)-cellulose column. After several washes with a solutioncontaining 10 mM Tris-HCl pH 7.4, 0.5% SDS and 0.5M NaCl, the polyA⁺ RNAwas collected by elution with 10 mM Tris-HCl pH 7.4 and 0.5% SDS andprecipitated with ethanol. Approximately 5 μg of the polyA⁺ mRNA wasused as template for reverse transcription primed with oligo(dT)primers. The reaction mixture (50 mM Tris-HCl pH 7.6, 10 mM DTT, 6 mMMgCl₂, 80 mM KCl, 0.2 mM each dNTP and 0.1 mg BSA/ml) was incubated for30 minutes at 37° C. with 500 units Murine MLV reverse transcriptase(BRL) and 75 units RNase inhibitor (Promega) in a volume of 100 μl.Another 200 units of reverse transcriptase were added and the reactionwas continued for 30 minutes. The mixture was extracted with chloroformand precipitated with ethanol in the presence of 0.25M ammonium acetate.This mixture of first strand cDNAs was used as template in a subsequentPolymerase Chain Reaction (PCR) to amplify the amdS cDNA. The genomicamdS sequence was used to design 2 synthetic oligonucleotides that wereused as primers in this PCR:

AB3100 (SEQ ID NO: 1)

5'-CTAATCTAGAATGCCTCAATCCTGAA-3' (an amdS-specific sequence fromnucleotide -3 to +16 preceded by an XbaI site and 4 additionalnucleotides).

AB3101 (SEQ ID NO: 2)

5'-GACAGTCGACAGCTATGGAGTCACCACA-3' (an amdS-specific sequence positioneddownstream of the amdS stopcodon from nucleotides 1911 to 1884 flankedby an additional SalI site).

The PCR reaction was performed using 10% of the cDNA mixture as templateand 0.1 μg of each of the oligos AB3100 SEQ ID NO: 1) and AB3101 (SEQ IDNO: 2) as primer. After denaturation (7 minutes at 100° C.) and additionof 1.3 units Taq-polymerase the reaction mixture was subjected to 25amplification cycles (each cycle: 2 minutes at 94° C., 2 minutes at 55°C. and 3 minutes at 72° C.). In the last cycle the extension step waslonger (7 min.) to allow synthesis of full-length fragments. Theobtained DNA fragment was digested with XbaI and SalI and subcloned intothe XbaI/SalI sites of pUC18. The resulting plasmid was designatedpamdS-1 (see FIG. 1). Restriction analysis of the plasmid pamdS-1confirmed the absence of introns and the correct fusion of exons in theamdS cDNA.

EXAMPLE 1 Marker Gene Free Deletion of an A.niger Gene by Using the amdSGene

In this example a genomic target gene in A.niger will be replaced bytransforming A.niger with a replacement vector which integrates into theA.niger genome via a double cross-over homologous recombination. Thereplacement vector comprises a DNA region homologous to the target locusinterrupted by a selectable marker gene flanked by DNA repeats.

In this example plasmid pGBDEL4L is used to delete the glaA codingregion and a (proximal) part of the glaA promoter region. This vectorcomprises a part of the A.niger glaA genomic locus, wherein the glaAcoding sequences as well as a part of the glaA promoter sequences arereplaced by the A.nidulans amdS gene under the control of A.nidulansgpdA promoter as selection marker flanked by 3'-untranslated glaAsequences as direct repeats. Transformation of A.niger with this vectordirects replacement of the glaA gene by the amdS marker gene. Byperforming the fluoracetamide counter-selection on these transformantsas described in the experimental procedures, the amdS marker gene willbe deleted properly by an internal recombination event between the3'glaA DNA repeats, resulting in a marker gene free ΔglaA recombinantstrain, possessing finally no foreign DNA sequences at all (for aschematic view, see FIG. 2).

Short description of the glaA gene replacement vector pGBDEL4L

The gene replacement vector pGBDEL4L contains 5'-part of the A.nigeramyloglucosidase (glaA) promoter region, a synthetic DNA sequence of 16bp providing stopcodons in all three reading frames, the A.nidulansacetamidase (amdS) gene under control of the A.nidulansglyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter, flanked atboth sides by 3' glaA non-coding sequences.

Construction pathway of pGBDEL4L

In order to obtain the final deletion vector pGBDEL4L several subclonesof the glaA locus were derived first. A schematic view is presented inFIG. 3. The glaA locus of A.niger was molecular cloned and describedpreviously (EP 0 463 706 A1). Plasmid pAB6-1 contains the entire glaAlocus from A.niger on a 15.5 kb HindIII fragment cloned in the HindIIIsite of pUC19 (Yanisch-Perron et al., Gene 33 (1985) 103-119, and isobtainable from e.g. Boehringer Mannheim, Germany). pAB6-1 was digestedwith EcoRI and the 1.8 kb EcoRI DNA fragment just upstream of the glaAgene was isolated by agarose gel electrophoresis and ligated into pUC19digested with EcoRI and subsequently transferred to E. coli andmolecular cloned. The resulting plasmid was designated pAB6-3 (FIG. 3A).To construct plasmid pAB6-4, which is another subclone of pAB6-1, pAB6-1was digested with HindIII and BglII. The 4.6 kb sized DNA fragmentcomprising the glaA promoter and a part of the glaA coding sequence wasisolated by agarose gel electrophoresis and ligated into pUC19 which wasdigested prior with HindIII and BamHI (FIG. 3B). As a result the BamHIas well as the BglII sites in pAB6-4 were destroyed appropriately bythis cloning procedure.

Subsequently, after digesting plasmid pAB6-4 with HindIII and EcoRI andfilling in the 5' sticky ends using E. coli DNA polymerase, the 1.8 kbglaA promoter DNA fragment was isolated by agarose gel electrophoresis,ligated into pAB6-3 which was partially digested with EcoRI and treatedwith E. coli DNA polymerase to generate blunt ends, the ligation mixturewas transferred to E. coli for molecular cloning. The derived plasmid(designated pAB6-31) contains a 3.6 kb glaA promoter fragment with adestroyed EcoRI site in the middle, but still possessing the EcoRI site(now unique in this DNA fragment) just upstream of the glaA ATGinitiation site (FIG. 4).

The A.nidulans amdS gene used herein is located on an approximately 4 kbsized EcoRI-KPnI fragment in plasmid pGW325 (Wernars et al., thesis(1986) Agricultural University, Wageningen, The Netherlands). ThisEcoRI-KpnI DNA fragment containing the amdS gene, flanked by its ownregulatory sequences, was molecular cloned into the appropriate sites ofpUC19 as described by Verdoes et al. (Transgenic Res. 2 pp 84-92, 1993)resulting in pAN4-1. pAN4-1 was digested with EcoRI and KpnI, the 4 kbsized DNA fragment containing the amdS gene was isolated by agarose gelelectrophoresis, ligated into pAB6-31 digested with EcoRI and KpnI andthe ligation mixture was transferred to E. coli for molecular cloning.The obtained plasmid was designated pAB6S (FIG. 5) and contains a 3.8 kbglaA promoter DNA fragment and the 4 kb amdS fragment.

Plasmid pAB6S was first partially digested with SalI, and ligated to thesynthetic derived oligonucleotide TN0001 (SEQ ID NO: 3) having thefollowing sequence:

TN0001 (SEQ ID NO: 3): 5' TCGATTAACTAGTTAA 3' and secondly digested withEcoRI. The DNA fragment comprising the pUC19, the glaA promoter and theamdS gene sequences was purified and isolated by agarose gelelectrophoresis. From plasmid pAB6-1, digested with SalI, the 2.2 kb 3'flanking glaA DNA fragment was isolated as well by agarose gelelectrophoresis and ligated to the above mentioned syntheticoligonucleotide, treated with T4 polynucleotide kinase, subsequentlydigested with EcoRI and ligated to the above mentioned DNA fragmentisolated of pAB6S. The DNA ligation mixture was transferred to E. coliand molecular cloned. The derived plasmid was designated pGBDEL1 and isshown in FIG. 6. By this procedure simultaneously the SalI restrictionsite was destroyed and stopcodons in all reading frames were introduced.

To obtain an approximately 1 kb large DNA fragment, containing 3' glaAnon-coding DNA sequences positioned just downstream the stop codon ofthe glaA gene and flanked by suitable restriction sites, a PCRamplification was performed. In this PCR amplification, the plasmidpAB6-1 was used as template and as primers two synthetical derivedoligonucleotides:

Oligo AB2154 (SEQ ID NO: 4)

5'AACCATAGGGTCGACTAGACAATCAATCCATTTCG 3' (a 3'glaA non-coding sequencejust downstream of the stopcodon) and

Oligo AB2155 (SEQ ID NO: 5)

5'GCTATTCGAAAGCTTATTCATCCGGAGATCCTGAT 3' (a 3'glaA non-coding sequencearound the EcoRI site approx. 1 kb downstream of the stopcodon).

The PCR was performed as described by Saiki et al. (Science 239,487-491, 1988) and according to the supplier of TAQ-polymerase (Cetus).Twenty five amplification cycles (each 2 minutes at 55° C.; 3 minutes at72° C. and 2 minutes at 94° C.) were performed in a DNA-amplifier(Perkin-Elmer/Cetus). The 1 kb amplified DNA fragment was digested withHindIII and SalI, purified by agarose gel electrophoresis, ethanolprecipitated and subsequently cloned into the HindIII and SalIrestriction sites of pGBDEL1. The thus obtained plasmid was designatedpGBDEL2 (FIGS. 7A,B).

To obtain the final glaA gene replacement vector pGBDEL4L, the amdSpromoter region in pGBDEL2 was exchanged by the stronger A.nidulans gpdApromoter. Fusion of the gpdA promoter sequence to the coding sequence ofthe amdS gene was performed by the Polymerase Chain Reaction (PCR)method. For this PCR fusion two different templates were used: plasmidpAN7-1 (Punt et al., Gene 56, 117-124, 1987) containing the E.coli hphgene under control of the A.nidulans gpdA promoter and the A.nidulanstrpC terminator and plasmid pAN4-1, containing the A.nidulans amdS geneunder control of its own regulatory sequences. As primers four syntheticoligonucleotides were used, possessing the following sequences:

Oligo AB 2977 (SEQ ID NO: 6)

5' TATCAGGAATTCGAGCTCTGTACAGTGACC 3' (a 5' gpdA promoter specific oligonucleotide, positioned at approximately 880 bp upstream of the ATGstartcodon of the E. coli hph gene)

Oligo AB2992 (SEQ ID NO: 7)

5' GCTTGAGCAGACATCACCATGCCTCAATCCTGGGAA 3'

Oligo AB2993 (SEQ ID NO: 8)

5' TTCCCAGGATTGAGGCATGGTGATGTCTGCTCAAGC 3' (both sequences arecomplementary to each other and contain 18 bp of the 3' end of the gpdApromoter and 18 bp of the 5' part of the amdS coding region)

Oligo AB2994 (SEQ ID NO: 9)

5' CTGATAGAATTCAGATCTGCAGCGGAGGCCTCTGTG 3' (an amdS specific sequencearound the BglII site approximately 175 bp downstream of the ATGinitiation codon)

To fuse the 880 bp gpdA promoter region to the amdS coding sequence twoseparate PCR's were carried out: the first amplification with pAN7-1 astemplate and the oligo nucleotides AB 2977 (SEQ ID NO: 6) and AB2993(SEQ ID NO: 8) as primers to amplify the 880 bp DNA fragment comprisingthe gpdA promoter flanked at the 3' border by 18 nucleotidescomplementary to the 5' end of the amdS gene, and the second PCRreaction with pAN4-1 as template and the oligo nucleotides AB2992 (SEQID NO: 7) and AB2994 (SEQ ID NO: 9) as primers to amplify a 200 bp sizedDNA fragment comprising the 5' part of the amdS gene flanked at the 5'border by 18 nucleotides complementary to the 3' end of the gpdApromoter. A schematic view of these amplifications is presented in FIG.8A. The two fragments generated were subsequently purified by agarosegel electrophoresis, ethanol precipitated and used as templates in athird PCR reaction with oligo nucleotides AB 2977 (SEQ ID NO: 6) andAB2994 (SEQ ID NO: 9) as primers. The resulting DNA fragment wasdigested with EcoRI, purified by agarose gel electrophoresis and ethanolprecipitation, and cloned into the EcoRI site of pTZ18R (United StatesBiochemicals). The resulting plasmid was designated pGBGLA24 (FIG. 8B).

To exchange the amdS promoter sequence in pGBDEL2 by the gpdA promotersequence, the approximately 1 kb sized EcoRI/BglII DNA fragment ofpGBGLA24 was isolated by agarose gel electrophoresis after digestionwith the appropriate restriction enzymes and ligated into the EcoRI andBglII sites of pGBDEL2. The resulting glaA gene replacement vector wasdesignated pGBDEL4L (FIG. 9).

Deletion of glaA promoter and coding sequences in A.niger

Prior to transformation of A.niger with pGBDEL4L, the E.coli sequenceswere removed by HindIII and XhoI digestion and agarose gelelectrophoresis. The A.niger strain CBS 513.88 (deposited Oct. 10, 1988)was transformed with either 2.5, 5 or 10 μg DNA fragment by proceduresas described in experimental procedures using acetamide as sole N-sourcein selective plates. Single A.niger transformants were purified severaltimes onto selective acetamide containing minimal plates. Spores ofindividual transformants were collected by growing for about 5 days at30° C. on 0.4% potato-dextrose (Oxoid, England) agar plates. Southernanalyses were performed to verify the presence of the truncated glaAlocus. High molecular weight DNA of several transformants was isolated,digested with BamHI and KpnI and subsequently fractionated byelectrophoresis on a 0.7% agarose gel. After transfer to nitrocellulosefilters, hybridization was performed according to standard proceduresusing two ³² P-labelled probes: a XhoI/SalI glaA promoter fragmentisolated from plasmid pAB6-4 (described above, FIG. 3A) and a proberecognizing endogenous xylanase sequences (European Patent Application.0 463 706 A). The results of only 4 transformants (#19, #23, #24, #41)and the control strain A.niger CBS 531.88 are shown as examples in FIG.10A. For a better understanding of this autoradiograph, a schematicpresentation is presented in FIG. 11 showing the size of the hybridizingfragments in intact and truncated glaA loci.

Characteristic for the intact glaA locus is a 3.5 kb hybridizingfragment in a BamHI digest and a 4.5 kb hybridizing fragment in a KpnIdigest (see FIG. 11A). In a truncated glaA locus, the 3.5 kb BamHIhybridizing fragment and the 4.5 kb KpnI hybridizing fragment are absentand replaced by a 5.5 kb BamHI hybridizing fragment and a 6.3 kb KpnIhybridizing fragment. In this example, as can be seen in FIG. 10A,transformant #19 shows the expected pattern of a truncated glaA locus(FIG. 11B). This transformant was designated GBA-102.

No replacement of the glaA gene had occurred in the other transformants.The poorly hybridizing bands: 4, 8 and 15 kb in the KpnI digest and 7and 12 kb in the BamHI digest, refer to the xylanase sequences asinternal control.

Removal of the amdS gene from A.niger GBA-102 by counter-selection onfluoracetamide containing plates

The amdS gene in the transformant A.niger GBA-102 was removed again asdescribed in the Experimental section. The removal of the amdS selectionmarker gene in only 2 surviving recombinant strains was verified bySouthern analysis of the chromosomal DNA. High molecular weight DNA wasisolated, digested with BamHI and KpnI and subsequently separated byelectrophoresis on a 0.7% agarose gel. Following transfer tonitrocellulose hybridization was performed according to standardprocedures using the probes described in the previous section. Aschematic presentation of the hybridizing fragments is shown in FIG.11C. The results of the Southern analyses are presented in FIG. 10B. Thepresence of a 5.2 kb hybridizing BamHI fragment and a 3.4 kb hybridizingKpnI fragment, with the concomitant loss of the 5.5 kb BamHI and the 6.3kb hybridizing KpnI fragments is specific for the absence of the amdSselection marker. The weaker hybridizing 7 and 12 kb fragments in aBamHI digest and the 4, 8 and 15 kb KpnI fragments again refer to theendogenous xylanase locus. Both strains show the expected pattern. Inthese recombinant strains, which were designated GBA-107 and GBA108, thepreferred glaA sequences are removed correctly and that possess finallyno selection marker gene at all. Both strains can be reused again todelete or insert other genes or DNA elements by using the same type ofvector.

EXAMPLE 2 Marker Gene Free Introduction of the glaA Gene Targeted at the3'glaA Non-coding Region of the Truncated glaA Locus in A.niger GBA-107

In this example the introduction of a gene into the genome of A.niger isdescribed by using approximately the same approach and procedures asdescribed in the previous example. Besides the desired gene or DNAelement the vector contains DNA sequences homologous to the host genometo target the vector at a predefined genomic locus of the host, by asingle cross-over event. This type of vector comprises a selectionmarker gene flanked by DNA repeats as well. The selection marker gene intransformants derived with this vector can be removed properly again byapplying the counter-selection procedure. As an example the introductionof a glaA gene copy is described which becomes integrated at thetruncated glaA locus in the recombinant ΔglaA A.niger GBA-107 strainderived in Example I (for a schematic drawing see FIG. 12)

Description of the glaA integration vector: pGBGLA30

The integration vector pGBGLA30 consists of the A.niger amyloglucosidase(glaA) gene under control of the native promoter and the A.nidulans amdSgene under control of the A.nidulans gpdA promoter flanked by 3'glaAnon-coding sequences to direct integration at the 3' glaA non-codingregion and to remove the amdS selection marker gene via thecounter-selection.

Construction of the integration vector

A 1.8 kb XhoI/EcoRI glaA promoter fragment from pAB6-1 (FIG. 13) wassubcloned into the SmaI and EcoRI sites of pTZ19R (United StatesBiochemicals). The protruding 5' end of the XhoI site of the glaApromoter fragment was filled in using the Klenow fragment of E.coli DNApolymerase I prior to cloning in pTZ19R. The SmaI site is destroyed andthe XhoI site is restored by this cloning procedure. The thus obtainedplasmid was designated pGBGLA5 (FIG. 13).

To introduce appropriate restriction sites (AatII, SnaBI, AsnI and NotI)and to destroy the XhoI site in the glaA promoter, the syntheticfragment consisting of the two oligonucleotides AB3657 (SEQ ID NO: 10)and AB3658 (SEQ ID NO: 11): ##STR1## was inserted into the HindIII andXhoI sites of pGBGLA5. The thus obtained plasmid was designated pGBGLA26(FIG. 14).

Next, the 3.4 kb EcoRI fragment from pAB6-1 containing the remaining 3'part of the glaA promoter, the glaA coding sequence and part of the 3'glaA non-coding sequence, was cloned into the EcoRI site of pGBGLA26.This new plasmid was designated pGBGLA27 (FIG. 15). This plasmid waspartially digested with EcoRI and the synthetic fragment consisting ofthe oligonucleotides AB3779 (SEQ ID NO: 12) and AB3780 (SEQ ID NO: 13):##STR2## was inserted into the EcoRI site at the end of the 3' glaAnon-coding sequence from the glaA gene. By this cloning step, the EcoRIsite was destroyed and an ApaI and XhoI restriction site wereintroduced. The resultant plasmid was designated pGBGLA42 (FIG. 16).

Amplification of the 2.2 kb 3' glaA non-coding sequences and concomitantadjustment of appropriate restriction sites was performed by thePolymerase Chain Reaction (PCR) method.

In these PCR reactions, plasmid pAB6-1 containing the entire glaA locuswas used as template and as primers four synthetic oligo nucleotideswere designed possessing the following sequence:

Oligo AB3448 (SEQ ID NO: 14)

5' GTGCGAGGTACCACAATCAATCCATTTCGC 3' (a 3' glaA non-coding specificsequence just downstream the stopcodon of the glaA gene)

Oligo AB3449 (SEQ ID NO: 15)

5' ATGGTTCAAGAACTCGGTAGCCTTTTCCTTGATTCT 3' (a 3' glaA non-codingspecific sequence around the KpnI site approx. 1 kb downstream of thestop codon)

Oligo AB3450 (SEQ ID NO: 16)

5' AGAATCAAGGAAAAGGCTACCGAGTTCTTGAACCAT 3' (a 3' glaA non-codingspecific sequence around the KpnI site approx. 1 kb downstream of thestop codon)

Oligo AB3520 (SEQ ID NO: 17)

5'ATCAATCAGAAGCTTTCTCTCGAGACGGGCATCGGAGTCCCG 3' (a 3' glaA non-codingspecific sequence approx. 2.2 kb downstream of the stopcodon)

To destroy the KpnI site approximately 1 kb downstream of the stop codonfrom the glaA gene and to alter the SalI site approximately 2.2 kbdownstream the stop codon from the glaA gene into a XhoI site twoseparate polymerase chain reactions were performed: the first reactionwith oligonucleotides AB3448 (SEQ ID NO: 14) and AB3449 (SEQ ID NO: 15)as primers to amplify an approximately 1 kb DNA fragment just downstreamthe stopcodon of the glaA gene, and the second reaction witholigonucleotides AB3450 (SEQ ID NO: 16) and AB3520 (SEQ ID NO: 17) asprimers to amplify an approximately 1.2 kb DNA fragment just downstreamthe KpnI site in the 3' glaA non-coding region both with pAB6-1 astemplate. A schematic view of these amplifications is presented in FIG.17A. The PCR was performed as described in example I. Twenty-fiveamplification cycles (each 1 minute at 55° C.; 1.5 minutes at 72° C. and1 minute at 94° C.) were carried out.

The two generated PCR DNA fragments were purified by agarose gelelectrophoresis and ethanol precipitation and subsequently used astemplate in the third PCR with oligonucleotides AB3448 (SEQ ID NO: 14)and AB3520 (SEQ ID NO: 17) as primers to generate the fusion fragment.Twenty-five amplification cycles (each: 2 minutes at 55° C.; 3 minutesat 72° C.; 2 minutes at 94° C.) were carried out in a DNA-amplifier(Perkin-Elmer/Cetus). The amplified DNA fragment was purified by agarosegel electrophoresis and ethanol precipitation and subsequently subclonedin the SmaI site of pTZ18R. The obtained plasmid was designated pGBGLA17(FIG. 17B).

To fuse this adjusted 3' glaA non-coding region to the amdS gene, a partof the amdS gene was subcloned from pGBDEL4L into pSP73 (Promega). Forthis construction, pGBDEL4L was digested with BglII and HindIII, the 3.4kb amdS/3'glaA non-coding fragment was isolated by agarose gelelectrophoresis and subcloned into the appropriate sites of pSP73(Promega). The resulting plasmid was designated pGBGLA21 (FIG. 18).

The approximately 1 kb sized 3' glaA non-coding region in this plasmidwas exchanged by the 2.2 kb 3' glaA non-coding region of pGBGLA17.pGBGLA17 and pGBGLA21 were digested with KpnI and HindIII. The 2.2 kb 3'glaA non-coding region DNA fragment from pGBGLA17 and the 4.9 kb DNAfragment of pGBGLA21 were isolated by agarose gel electrophoresis,ligated and subsequently molecular cloned by transferring the ligationmixture to E. coli. The thus derived plasmid was designated pGBGLA22(FIG. 19).

The amdS gene with the extended 3'glaA non-coding region was completedwith the gpdA promoter and fused to the remaining part of the amdS gene.pGBGLA22 was digested with BglII and HindIII, the 4.4 kb amdS/3'glaAnon-coding region DNA fragment isolated by agarose gel electrophoresis,subsequently ligated with plasmid pGBGLA24 digested with BglII andHindIII and transferred to E. coli. The thus derived plasmid wasdesignated pGBGLA25 (FIG. 20).

pGBGLA25 was partially digested with EcoRI and in the EcoRI site of thegpdA promoter the synthetic fragment consisting of the twooligonucleotides AB3781 (SEQ ID NO: 18) and AB3782 (SEQ ID NO: 19):##STR3## was inserted. This new plasmid was designated pGBGLA43 (FIG.21). Due to this cloning step, the EcoRI restriction site just in frontof the gpdA promoter was destroyed by the introduction of an ApaIrestriction site.

The plasmid pGBGLA43 was digested with ApaI and XhoI, and the 5.3 kb DNAfragment comprising the gpdA promoter/amdS gene/3'glaA non-coding regionwas isolated by agarose gel electrophoresis, subsequently ligated withpGBGLA42 digested with ApaI and XhoI, and transferred to E.coli. Thederived plasmid was designated pGBGLA28 (FIG. 22).

Prior to cloning, the 3'glaA non-coding region DNA fragment (positionedat approximately 2.2 kb downstream the stop codon of the glaA gene,designated 3"glaA non-coding region), was amplified and provided withsuitable restriction sites using the PCR method.

For this PCR reaction, the plasmid pAB6-1 was used as template and asprimers two synthetic oligonucleotides were designed possessing thefollowing sequence:

Oligo AB3746 (SEQ ID NO: 20)

5' TGACCAATAAAGCTTCTCGAGTAGCAAGAAGACCCAGTCAATC 3' (a partly 3"glaAnon-coding specific sequence around the SalI site positioned at about2.2 kb downstream the stop codon of the glaA gene)

Oligo AB3747 (SEQ ID NO: 21)

5'CTACAAACGGCCACGCTGGAGATCCGCCGGCGTTCGAAATAACCAGT3' (a partly 3"glaAnon-coding specific sequence around the XhoI site located at about 4.4kb downstream the stop codon of the glaA gene)

Twenty-five amplification cycles (each: 1 minute 55° C.; 1.5 minutes 72°C.; 1 minute 94° C.) were carried out in a DNA-amplifier(Perkin-Elmer/Cetus). A schematic representation of this amplificationis shown in FIG. 23A. The thus obtained DNA fragment was digested withHindIII, purified by agarose gel electrophoresis and ethanolprecipitation and subcloned in both orientations into the HindIII siteof pTZ19R. The resulting plasmids were designated pGBGLA29A andpGBGLA29B (FIG. 23).

The final step comprises the insertion of the 3"glaA non-coding sequencefrom pGBGLA29A into the plasmid pGBGLA28. To achieve this, pGBGLA29A wasdigested with HindIII and NotI. The 2.2 kb sized 3'glaA non-codingregion fragment was isolated by agarose gel electrophoresis,subsequently ligated to pGBGLA28 digested with HindIII and NotI andtransferred to E. coli. The derived integration vector was designatedpGBGLA30 (FIG. 24).

Transformation of A.niger GBA-107 with the integration vector pGBGLA30

Prior to transformation, E.coli sequences were removed from theintegration vector pGBGLA30 by XhoI digestion and agarose gelelectrophoresis. The A.niger strain GBA-107 was transformed with either5 or 10 μg DNA fragment by procedures as described in the experimentalsection. Single A.niger transformants were purified several times onselective acetamide containing plates. Spores of individualtransformants were collected following growth for about 5 days at 30° C.on 0.4% potato dextrose agar (Oxoid, England) plates. Southern analyseswere performed to verify whether integration into the 3' glaA non codingregion of the endogenous truncated glaA locus had occurred. Highmolecular weight DNA of several transformants was isolated, digestedwith either KpnI, or BglII and subsequently fractionated byelectrophoresis on a 0.7% agarose gel. After transfer to nitrocellulosefilters, hybridization was performed according to standard procedures.As probe a ³² P-labelled approx. 0.7 kb XhoI/SalI glaA promoter fragmentisolated from plasmid pAB6-4 (described in example 1) was used. Theresults of only 3 transformants (#107-5, #107-9 and #107-7) and thereference strain A.niger GBA107 and its ancestor A.niger CBS 531.88 areshown as example in FIG. 25. For a better understanding of theautoradiograph, a schematic presentation is given in FIGS. 26A,B,Cshowing the sizes of the hybridizing fragments of the intact glaA locus,the truncated glaA locus and of the truncated glaA locus with a singlepGBGLA30 copy integrated into the predefined 3' glaA non-coding region.

Characteristic for the intact glaA locus is a 4.5 kb hybridizingfragment in a KpnI digest and a 10 kb hybridizing fragment in a BglIIdigest. Characteristic for the truncated glaA locus of A.niger GBA-107is a 3.4 kb hybridizing fragment in a KpnI digest and a 13 kbhybridizing fragment in a BglII digest. In case of integration of thepGBGLA30 vector into the 3' region of the truncated glaA locus, in aKpnI digest an additional 6.7 kb hybridizing fragment is expectedbesides the 3.4 kb hybridizing fragment and in a BglII digest the 13 kbhybridizing fragment is absent and replaced by a 14.5 kb hybridizingfragment. As can be seen in FIG. 25, transformants #107-5 and #107-9show the expected hybridization pattern of a single pGBGLA30 copyintegrated into the predefined 3' non-coding region of the truncatedglaA locus. The hybridization pattern of transformant #107-7 indicatesintegration of the pGBGLA30 copy elsewhere into the genome of A.nigerGBA-107. The transformants with the correctly integrated pGBGLA30 copywere designated GBA-119 and GBA-122 and were used to remove subsequentlythe amdS selection marker gene properly.

Removal of the amdS selection marker gene from A.niger GBA-119 andGBA-122 by counter-selection on fluoracetamide containing plates

The amdS selection marker gene in the transformants A.niger GBA-119 andGBA-122 was removed again as described in the experimental section. Theremoval of the amdS selection marker gene in several survivingrecombinant strains was verified by Southern analysis of the chromosomalDNA. High molecular weight DNA was isolated, digested either with KpnIor BglII and subsequently separated by electrophoresis on a 0.7% agarosegel. Following transfer to nitrocellulose, hybridization was performedaccording to standard procedures. As probe the ³² P labelled 2.2 kbHindIII/NotI 3"glaA non-coding fragment isolated from plasmid pGBGLA29A(described previously, FIG. 24) was used.

A schematic presentation of the hybridizing fragments is shown in FIG.26. The results of only 3 surviving recombinant strains from A.nigerGBA-119 (#AG5-5, #AG5-6 and #AG5-7) as well as 3 surviving recombinantstrains from A.niger GBA-122 (#AG9-1, #AG9-2 and #AG9-4) and thereference strains A.niger CBS 531.88 and A.niger GBA-107 are shown inFIGS. 27A,B.

In strain A.niger CBS 531.88 a 6.9 kb hybridizing fragment is present ina KpnI digest and a 6.9 kb hybridizing fragment in a BglII digest. Inthe A.niger GBA-107 strain a 6.9 kb hybridizing fragment is present in aKpnI digest and a 13 kb hybridizing fragment in a BglII digest. In theA.niger strains GBA-119 and GBA-122 with a single pGBGLA30 copyintegrated into the 3' glaA non-coding region an 8 kb and a 6.7 kbhybridizing band are present in a KpnI digest and a 14.5 kb and a 7.6 kbhybridizing band are present in a BglII digest.

Specific for correct removal of the amdS selection marker gene is thepresence of a 6.7 kb and a 8.5 kb hybridizing fragment in a KpnI digestand concomitant loss of the 8 kb hybridizing fragment. In a BglIIdigest, a 14.5 kb and a 6.9 kb hybridizing fragment with concomitantloss of the 7.6 kb hybridizing fragment is specific for the absence ofthe amdS selection marker gene. As can be seen in FIG. 27, strains#AG5-7, #AG5-5, #AG9-1 and #AG9-4 show the expected hybridizing patternof the correctly removed amdS selection marker gene. These strains weredesignated GBA-120, GBA-121, GBA-123 and GBA-124 respectively. Thehybridizing patterns of strains #AG5-6 and #AG9-2 indicate loss of theentire pGBGLA30 copy resulting in the parental A.niger GBA-107 strainwith only a truncated glaA locus.

Strains A.niger GBA-120, GBA-121, GBA-121 and GBA-124 were tested inshake flask fermentations for the ability to produce glucoamylase. Asreference strains A.niger CBS 531.88, GBA-107, GBA-119 and GBA-122 weretested. Shake flask fermentations and the glucoamylase assay wereperformed as described in the experimental section. In the strainsGBA-119 till GBA-124 levels varying between 150-200 U/ml could bemeasured. These glucoamylase levels were to be expected and comparableto levels obtained with the parental untransformed wild-type strainA.niger CBS 531.88.

EXAMPLE 3 Marker Gene Free Introduction of the Phytase Gene Targeted atthe 3'glaA Non-coding Region of the Truncated glaA Locus in A.nigerGBA-107

In this example describes the introduction of a gene into the genome ofA.niger by using approximately the same approach and procedures asdescribed in the previous example. The main difference is that the geneof interest and the selection marker gene are located on two separatevectors and that these vectors are co-transformed to A.niger. Besidesthe gene of interest or the marker gene, the vectors contain DNAsequences homologous to the host genome to target the vectors at apredefined genomic locus of the host, by a single cross-over event. Byperforming the fluoracetamide counter-selection on these(co)-transformants (as described in the experimental procedures), theamdS marker gene will be deleted properly by an internal recombinationevent between the DNA repeats that are created by integration via asingle cross-over event.

Description of the vectors used for co-transformation

The vector with the gene of interest pGBGLA53 consists of the A.ficuumphytase gene under control of the A.niger glucoamylase (glaA) promoterflanked by 3'glaA non-coding sequences to direct integration at the3'glaA non-coding region. The vector with the selection marker genepGBGLA50 consists of the A.nidulans amdS gene under control of theA.nidulans gpdA promoter flanked by 3'glaA non-coding sequences todirect integration at the 3'glaA non-coding region.

Construction pathway of pGBGLA50

The construction of pGBGLA50 comprises one cloning step. PlasmidpGBGLA29A was digested with HindIII and the sticky ends were filled inusing the Klenow fragment of E.coli DNA polymerase. Next, the 2.2 kb3"glaA non-coding region fragment was isolated by agarosegel-electrophoresis, subsequently ligated into pGBGLA43 digested withApaI and treated with T4 DNA polymerase to generate blunt ends, andtransferred to E.coli. The derived plasmid with the 3"glaA non-codingregion DNA fragment in the correct orientation was designated pGBGLA50(FIG. 28).

Construction pathway of pGBGLA53

The first step in the construction pathway of pGBGLA53 is the subcloningof two fragments, comprising the glaA promoter fused to almost entirecoding sequence of the A.ficuum phytase gene. To achieve this, plasmidpGBGLA42 was digested with HindIII and EcoRI and the 1.8 kbHindIII/EcoRI 5'glaA promoter fragment was isolated by agarosegel-electrophoresis. Plasmid pFYT3 (European Patent Application 0 420358 A1) was digested with EcoRI and BglII and the 1.6 kb EcoRI/BglIIfragment comprising the 3'part of the glaA promoter fused to the 5' partof the phytase gene was isolated by agarose gel-electrophoresis andligated together with the 1.8 kb HindIII/EcoRI 5'glaA promoter fragmentisolated from pGBGLA42 into the HindIII and BglII sites of pSp73(Promega). The resulting plasmid was designated pGBGLA49 (FIG. 29).

The next step is the cloning of a 3'glaA non-coding region DNA fragmentinto pGBGLA49. Prior to cloning, this 3'glaA non-coding region DNAfragment (positioned at approximately 2.2 kb downstream the stop codonof the glaA gene) was amplified and provided with suitable restrictionsites using the PCR method.

For this PCR reaction, the plasmid pAB6-1 was used as template and asprimers two synthetic oligonucleotides with the following sequence weredesigned:

Oligo AB4234 (SEQ ID NO: 22)

5' GAAGACCCAGTCAAGCTTGCATGAGC 3' (a 3'glaA non-coding sequence locatedapproximately 2.2 kb downstream the stopcodon of the glaA gene)

Oligo AB 4235 (SEQ ID NO: 23)

5'TGACCAATTAAGCTTGCGGCCGCTCGAGGTCGCACCGGCAAAC 3' (a 3'glaA non-codingsequence located approximately 4.4 kb downstream the stopcodon of theglaA gene)

Twenty-five amplification cycles (each: 1 minute 94° C.; 1 minute 55°C.; 1.5 minutes 72° C.) were carried out in a DNA-amplifier(Perkin-Elmer). A schematic representation of this amplification isshown in FIG. 30A. The thus obtained fragment was digested with HindIII,purified by agarose gel-electrophoresis and subcloned into the HindIIIsite of pTZ19R. The resulting plasmid was designated pGBGLA47 (FIG. 30).

Plasmid pGBGLA47 was digested with HindIII en NotI, the 2.2 kb 3"glaAnon-coding DNA fragment was isolated by agarose gel-electrophoresis andcloned into the HindIII and NotI sites of pGBGLA49. The resultingplasmid was designated pGBGLA51 (FIG. 31).

The last step in the construction pathway of pGBGLA53 is the cloning ofthe DNA fragment comprising the remaining part of the phytase codingsequence fused to the 3'glaA non-coding DNA fragment located justdownstream the stop codon of the glaA gene. Prior to cloning, theremaining part of the phytase gene and the 3'glaA non-coding DNAfragment located just downstream the stopcodon of the glaA gene werefused and provided with suitable restriction sites using the PCR method.In the PCR, plasmid pAB6-1 was used as template and as primers twosynthetic oligonucleotides were used, having the following sequences:

Oligo AB4236 (SEQ ID NO: 24)

5' TGACCAATAAAGCTTAGATCTGGGGGTGATTGGGCGGAGTGTTTTGCTTAGACAATCAATCCATTTCGC 3' (36 bp of the phytase coding sequence, startingat the BglII site until the stopcodon fused to the 3'glaA non-codingregion, starting just downstream the stopcodon of the glaA gene)

Oligo AB4233 (SEQ ID NO: 25)

5' TGACCAATAGATCTAAGCTTGACTGGGTCTTCTTGC 3' (a 3'glaA non-coding sequencelocated approximately 2.2 kb downstream the stopcodon of the glaA gene)

Twenty-five amplification cycles (each: 1 minute 94° C.; 1 minute 55°C.; 1.5 minutes 72° C.) were carried out in a DNA-amplifier(Perkin-Elmer). A schematic representation of this amplification isshown in FIG. 32A. The thus obtained fragment was digested with HindIII,purified by agarose gel-electrophoresis and subcloned in bothorientations into the HindIII site of pTZ19R. The resulting plasmidswere designated pGBGLA48 and pGBGLA52 (FIG. 32B).

Plasmid pGBGLA52 was digested with BglII and partially digested withBamHI, the 2.2 kb phytase/3'glaA non-coding DNA fragment was isolated byagarose gel-electrophoresis and cloned into the BglII site of pGBGLA51.The derived plasmid with the 2.2 kb phytase/3'glaA non-coding DNAfragment in the correct orientation was designated pGBGLA53 (FIG. 33).

Transformation of A.niger GBA-107 with the vectors pGBGLA50 and pGBGLA53

Prior to transformation, E.coli sequences were removed from pGBGLA50 andpGBGLA53 by respectively XhoI or HindIII digestion followed by agarosegel-electrophoresis. The A.niger GBA-107 strain was transformed withrespectively 1 μg pGBGLA50 fragment plus 1 μg pGBGLA53 fragment, 1 μgpGBGLA50 fragment plus 5 μg pGBGLA53 fragment, or 1 μg pGBGLA50 fragmentplus 10 μg pGBGLA53 fragment using the transformation proceduredescribed in the experimental section.

Single transformants were isolated, purified and Southern analysis wasperformed, using the same digests and probes as described in example 2,to verify integration of both pGBGLA50 and pGBGLA53. In about 10-20% ofthe analyzed transformants both pGBGLA50 and pGBGLA53 were integratedinto the genome of the A.niger GBA-107 host strain. The transformantshowing the correct integration pattern of a single copy pGBGLA50 and asingle copy pGBGLA53, both integrated at the predefined 3'glaAnon-coding region of the truncated glaA locus was used to removesubsequently the amdS selection marker gene.

Removal of the amdS marker gene by counter-selection on fluoracetamidecontaining plates

By performing the fluoracetamide counter-selection (as described in theexperimental procedures), the amdS marker gene was deleted by aninternal recombination event between the DNA repeats that were createdby integration via a single cross-over event (i.e. the 3'glaA non-codingsequences). Proper removal of only the amdS marker gene was verified bySouthern analysis using the same digests and probes as in example 2.

EXAMPLE 4 Marker Gene Free Introduction of the glaA Gene and the PhytaseGene in A.oryzae

This example describes the marker gene free introduction of the glaAgene or the phytase gene in A.oryzae NRRL3485. A.oryzae NRRL3485 wastransformed as described in the experimental section using the samevectors and approach as described in examples 2 and 3. Singletransformants were isolated, purified and Southern analysis ofchromosomal DNA of several transformants was performed to verifyintegrations of respectively the pGBGLA30 vector or the pGBGLA50 andpGBGLA53 vectors. In the Southern analysis, the same digests and probeswere used as described in example 2.

Removal of the amdS gene by counter-selection on fluoracetamidecontaining plates

In case of integration of the pGBGLA30 vector, a transformant with asingle copy of the pGBGLA30 integrated into the genome of the hoststrain A.oryzae NRRL3485 was used to remove the amdS gene properly. Thecounter-selection on fluoracetamide containing plates was performed asdescribed in the experimental section. Correct removal of the amdS genewas verified by Southern analysis of chromosomal DNA of severalfluoracetamide resistant strains. The same digests and probes were usedas described in Example 2.

In case of co-transformation of the pGBGLA50 and pGBGLA53 vector, atransformant with a single copy of both pGBGLA50 and pGBGLA53 integratedinto the host genome was used to remove the amdS marker gene properly.The counter-selection using fluoracetamide plates was performed asdescribed in the experimental section. Correct removal of the amdSmarker gene (e.g. the pGBGLA50 vector) was verified by Southern analysisof chromosomal DNA of several fluoracetamide resistant strains using thesame digests and probes as described in example 2.

EXAMPLE 5 Marker Gene Free Introduction of the glaA Gene and the PhytaseGene in T.reesei

This example describes the marker gene free introduction of the glaAgene or the phytase gene in Trichoderma reesei strain QM9414 (ATCC26921). T.reesei QM9414 was transformed as described in the experimentalsection using the same vectors and approach as described in examples 2and 3. Single transformants were isolated, purified and Southernanalysis of chromosomal DNA of several transformants was performed toverify whether integration of respectively the pGBGLA30 vector or thepGBGLA50 and pGBGLA53 vectors. In the Southern analysis, the samedigests and probes were used as described in example 2.

Removal of the amdS gene by counter-selection on fluoracetamidecontaining plates

In case of integration of the pGBGLA30 vector, a transformant with asingle copy of the pGBGLA30 integrated into the genome of the hoststrain T.reesei QM9414 was used to remove the amdS gene properly. Thecounter-selection on fluoracetamide containing plates was performed asdescribed in the experimental section. Correct removal of the amdS genewas verified by Southern analysis of chromosomal DNA of severalfluoracetamide resistant strains.

In case of co-transformation of the pGBGLA50 and pGBGLA53 vector, atransformant with a single copy of both pGBGLA50 and pGBGLA53 integratedinto the host genome was used to remove the amdS marker gene properly.The counter-selection using fluoracetamide plates was performed asdescribed in the experimental section. Correct removal of the amdSmarker gene (e.g. the pGBGLA50 vector) was verified by Southern analysison chromosomal DNA of several fluoracetamide resistant strains using thesame digests and probes as described in example 2.

EXAMPLE 6 Marker Gene Free Introduction into P.chrysogenum of aP.chrysogenum Gene by Co-transformation using the amdS-gene as aSelection Marker

In this example the marker gene free introduction of a gene into thegenome of P.chrysogenum by co-transformation is described.

In the co-transformation procedure, 2 different pieces of DNA areoffered to the protoplasts, one of them being the amdS-selection marker,on the presence of which the first transformant selection takes place,as described in the experimental section, the second being another pieceof DNA of interest, e.g. encoding a particular enzyme of interest. In acertain number of transformants both pieces of DNA will integrate intothe chromosomes and will be stably maintained and expressed.

The amdS-selection marker gene can then be removed selectively frompurified transformants by applying the counter-selection procedure asdescribed in the experimental section, while the second piece of DNAwill remain stably integrated into the chromosomes of the transformant.As an example to illustrate the general applicability of the method thestable, marker gene free introduction of a niaD-gene is described whichenables a niaD⁻ -host to grow on nitrate as sole nitrogen-source.

Host for this co-transformation is a P.chrysogenum niaD⁻ -strain whichlacks nitrate reductase and therefore is unable to grow on platescontaining nitrate as sole nitrogen source. These strains can be easilyobtained by well known procedures (Gouka et al., Journal ofBiotechnology 20(1991), 189-200 and references there in).

During the co-transformation (procedure described in experimentalsection), two pieces of DNA are simultaneously offered to theprotoplasts: the 7.6 kb EcoRI restriction fragment from pGBGLA28containing the amdS selection marker gene and the 6.5 kb EcoRIrestriction fragment from pPC1-1, containing the P.chrysogenumniaD-gene. Prior to transformation, both fragments have been separatedfrom E.coli vector sequences by agarose gel-electrophoresis and purifiedfrom agarose gel by electro-elution. The first selection oftransformants took place on selective plates containing acetamide assole nitrogen source as described in the experimental section.

Among the transformants, co-transformants are found by replica platingspores of purified transformants to plates containing nitrate as solenitrogen source.

Typically about 20-60% of the replica plated transformants were able togrow on this medium, indicating that in these transformants not only theamdS selection marker gene but also the niaD-gene has integrated intothe genome and is expressed.

Removal of the amdS gene by counter-selection on fluoracetamidecontaining plates

The amdS selection marker gene is subsequently removed from theco-transformants by counter-selection on fluor-acetamide.

For direct selection on the amdS⁻ /niaD⁺ -phenotype the medium usedcontained 10 mM fluor-acetamide. Spores were plated at a density of 10⁴spores per plate. After 5-7 days of incubation at 25° C.,fluor-acetamide resistant colonies could be identified as solid coloniesclearly distinct from the faint background. The niaD⁺ -phenotype of therecombinants is demonstrated by their growth on thefluoracetamide-medium containing nitrate as sole nitrogen source. TheamdS⁻ -phenotype of the recombinants was confirmed by lack of growth ofthe recombinants on plates containing acetamide as sole nitrogen source.Typically, 0.1-2% of the original number of plated spores exhibited thedesired phenotype.

Southern analysis on chromosomal DNA form several fluoracetamideresistant strains confirmed that the amdS selection marker gene wasremoved from the P.chrysogenum genome.

EXAMPLE 7 Test of the amdS-minus Phenotype of the Yeast Kluyveromyceslactis

A prerequisite for the use of the amdS selection system in K.lactis isthat this yeast does not contain any acetamidase activity. To test thiswe have plated the K.lactis strains CBS 683 and CBS 2360 on thefollowing 3 different solid media:

I Yeast Carbon Base (YCB, Difco), containing all the essentialnutritives and vitamins except a nitrogen-source.

II YCB supplemented with 5 mM acetamide.

III YCB supplemented with 0.1% (w/v) NH₄ (SO₄)₂.

All 3 media contained 1.2% (w/v) Oxoid agar (Agar No. 1) and 30 mMSodium Phosphate buffer at pH 7.0. Difco YCB was used at 1.17% (w/v).

Full grown K.lactis colonies were only observed on medium III,containing ammonium as nitrogen source. In plates withoutnitrogen-source or with acetamide as sole nitrogen-source no growth or,occasionally slight background growth was observed, which is most likelycaused by trace amounts of nitrogen contaminating the agar or othermedium components. We conclude that both K.lactis strains lacksufficient acetamidase activity to sustain growth on acetamide as solenitrogen source. This should allow for the A.nidulans amdS gene to beused as selection marker in the yeast K.lactis.

EXAMPLE 8 Construction of Plasmids for use of the amdS Gene in Yeasts

Construction of pGBamdS1

We have previously used pGBHSA20 for the expression of human serumalbumin (HSA) in K.lactis (Swinkels et al. 1993, Antonie van Leeuwenhoek64, 187-201). In pGBHSA20 the HSA cDNA is driven from the K.lactis LAC4promoter (FIG. 34 for the physical map of the plasmid pGBHSA20). At the3'-end the HSA cDNA is flanked by LAC4 terminator sequences. Forselection of transformants pGBHSA20 contains the Tn5 phosphotransferasegene which confers resistance to the antibiotic G418 (Geneticin, BRL)(Reiss et al. (1984) EMBO J. 3, 3317-3322) driven by the S.cerevisiaeADH1 promoter (Bennetzen and Hall (1982) J. Biol. Chem. 257, 3018-3025).In the unique SstII site of the LAC4 promoter pGBHSA20 contains theE.coli vector pTZ19R which is used for amplification in E.coli. Prior totransformation to K.lactis the pTZ19R sequences are removed frompGBHSA20 by SstII digestion and agarose gel purification. Transformationof pGBHSA20 linearized in the SstII site of the LAC4 promoter toK.lactis results in integration into the genomic LAC4 promoter byhomologous recombination. pGBamdS1 is derived from pGBHSA20 bysubstitution of the HSA cDNA for the amdS cDNA from pamdS1. Using PCR,SalI sites were introduced at the 5' and 3' ends of the amdS cDNA. Inthis PCR pamdS1 was used as template and oligo's AB3514 (SEQ ID NO: 26)and AB3515 (SEQ ID NO: 27) were used as primers.

Oligo AB3514 (SEQ ID NO: 26)

5'-CTGCGAATTCGTCGACATGCCTCAATCCTGGG-3' (an 5'end amdS-specific sequencewith the introduced SalI site)

Oligo AB3515 (SEQ ID NO: 27)

5'-GGCAGTCTAGAGTCGACCTATGGAGTCACCACATTTC-3' (an 3' end amdS-specificsequence with the introduced SalI site).

The PCR fragment thus obtained was digested with SalI and cloned intothe SalI/XhoI sites of pGBHSA20. Several clones were obtained containingeither of the 2 possible orientations of the amdS cDNA as judged byrestriction analysis. One of the clones with the amdS cDNA in thecorrect orientation is pGBamdS1, the physical map of which is shown inFIG. 34.

Construction of pGBamdS3

By heterologous hybridization using a probe derived from theS.cerevisiae elongation factor 1-α gene (EF1-α; Nagata et al. (1984)EMBO J. 3, 1825-1830), we have isolated a genomic clone containing theK.lactis homologue of the EF1-α gene, which we call KlEF1. In thisexample we have used a 813 bp fragment containing the KlEF1 promoter toexpress the amdS cDNA in K.lactis. Using oligonucleotides AB3701 (SEQ IDNO: 28) and AB3700 (SEQ ID NO: 29), this fragment was amplified in a PCRusing genomic DNA from K.lactis strain CBS 683 as template. AB3700 (SEQID NO: 29) is designed such that it contains 21 nucleotides of the KlEFpromoter and 38 nucleotides upstream the ATG initiation codon of theamdS gene. The sequence of AB3701 (SEQ ID NO: 28) and AB3700 (SEQ ID NO:29) is as shown:

Oligo AB3701 (SEQ ID NO: 28)

5'-CTGCGAATTCGTCGACACTAGTGGTACCATTATAGCCATAGGACAGCAAG 3' (a 5'KlEF1-specific promoter sequence with the additional restriction sitesEcoRI, SalI, SpeI and KpnI at the 5' end of the promoter)

Oligo AB3700 (SEQ ID NO: 29)

5'-GCTCTAGAGCGCGCTTATCAGCTTCCAGTTCTTCCCAGGATTGAGGCATTTTTAATGTTACTTCTCTTGC-3'(3' KlEF1-specific promoter sequence fused to the 5'-sequences of theamdS cDNA with the restriction sites BssH2 and additional site XbaI).

The PCR was performed using standard conditions and the PCR-fragmentobtained was digested with EcoRI and XbaI and subcloned into EcoRI/XbaIdigested pTZ19R. The physical map of the resulting plasmid pTZKlEF1 isshown in FIG. 35. The remaining part of the amdS cDNA as well as part ofthe LAC4 terminator sequences were obtained from pGBamdS1 by digestionwith BssH2 and SphI. This BssH2-SphI fragment was cloned into the BssH2and SphI digested pTZKlEF1 and the resulting plasmid was designatedpGBamdS2 (FIG. 35). For the final step in the construction of pGBamdS3,both pGBamdS2 and pTY75LAC4 (Das and Hollenberg (1982) Current Genetics6, 123-128) were digested were digested with SphI and HindIII. The 5.7kb DNA fragment from pGBamdS2 and the 1.2 kb DNA fragment frompTY75LAC4, which contains the remaining LAC4 terminator sequences, werepurified from agarose gels after fractionation and subsequently ligatedand used to transform E.coli. The resulting expression vector, in whichthe amdS cDNA is driven from the K.lactis KlEF1 promoter, was designatedpGBamdS3 (FIG. 36).

Construction of pGBamdS5

Fusion of the S.cerevisiae alcohol dehydrogenase I (ADH1) promoter tothe amdS cDNA was performed in a PCR using pGBHSA20 as template. One ofthe primers (AB3703; SEQ ID NO: 31) contains sequences complimentary tothe 3'-end of the ADH1-promoter sequence which are fused to sequences ofthe amdS cDNA. The other primer (AB3702; SEQ ID NO: 30) contains the5'-end of the ADH1 promoter:

Oligo AB3702 (SEQ ID NO: 30)

5'-CTGCGAATTCGTCGACACTAGTGGTACCATCCTTTTGTTGTTTCCGGGTG-3' (a 5'ADH1-specific promoter sequence with the additional restriction sitesEcoRI, SalI, SpeI and KpnI at the 5' end of the promoter).

Oligo AB3703 (SEQ ID NO: 31)

5'-GCTCTAGAGCGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATTGTATATGAGATAGTTGATTG-3'(a 3' ADH1-specific promoter sequence fused to the 5' amdS sequence withadditional restriction sites BssH2 and XbaI).

The PCR reaction was performed using a "touchdown" protocol (Don et al.,(1991) Nucleic Acids Res. 19, 4008). The reaction mixtures weresubjected to 30 amplification cycles, while the annealing temperaturewas decreased 1° C. every two cycles, starting 55° C. down to a"touchdown" at 40° C., at which temperature 10 more cycles were carriedout (cycles: 2' at 94° C., 2' annealing, 3' at 72° C.). The PCR-fragmentobtained was digested with EcoRI and XbaI and subcloned into pTZ19R. Theresulting plasmid pTZs.c.ADH1 is shown in FIG. 37. pTZs.c.ADH1 andpGBamdS3 were digested with KPnI and BssH2. The 6.8 kb fragment frompGBamdS3 and the 750 bp fragment from pTZs.c.ADH1 were purified by gelelectrophoresis, ligated and used to transform E.coli JM109. Theresulting expression vector was designated pGBamdS5 (FIG. 37).

Construction of pGBamdS6

Plasmid pGBamdS3 contains the amdS cDNA under control of the KlEF1promoter and flanked at the 3' end by 1.5 kb of LAC4 terminatorsequences (FIG. 36). pGBamdS6 is constructed by cloning a fragment whichcontains a fusion of the LAC4 promoter and terminator sequences upstreamof the amdS expression-cassette in pGBamdS3 (FIG. 38). In order to fusethe LAC4 promoter and terminator sequences we have first constructedpPTLAC4 (FIG. 39). Using a PCR, additional restriction sites areintroduced at the 5' and 3' end of a 600 bp LAC4 terminator fragment. Inthe PCR K.lactis CBS 683 chromosomal DNA was used as template andoligonucleotides AB3704 (SEQ ID NO: 32) and AB3705 (SEQ ID NO: 33) wereused as primers:

Oligo AB3704 (SEQ ID NO: 32)

5'-GCTCTAGAAGTCGACACTAGTCTGCTACGTACTCGAGAATTTATACTTAGATAAG-3' (a LAC4terminator-specific sequence starting at the LAC4 stop codon with theadditional restriction sites XbaI, SalI, SpeI, SnaBI and XhoI).

Oligo AB3705 (SEQ ID NO: 33)

5'-TGCTCTAGATCTCAAGCCACAATTC-3' (3' LAC4 terminator-specific sequencewith the additional restriction site XbaI).

The PCR was performed using standard conditions and the resulting DNAfragment was digested with XbaI and subcloned into the XbaI site ofpTZ19R to give pTLAC4 (FIG. 39). The LAC4 promoter sequence is obtainedby digestion of pKS105 van den Berg et al. (1990) Bio/Technology 8,135-139) with XbaI and SnaBI. The XbaI-SnaBI LAC4 promoter fragment wascloned into the SpeI/SnaBI sites of pTLAC4 and designated pPTLAC4 (FIG.39). For the final step in the construction of pGBamdS6, the plasmidpPTLAC4 was digested with XbaI. The 4.1 kb DNA fragment from pPTLAC4 waspurified by gel-electrophoresis and cloned into SpeI site of pGBamdS3.The obtained gene-replacement vector was designated pGBamdS6 (FIG. 38).

Construction of pGBamdS8

pGBamdS7 was constructed by cloning a fragment, which contains part ofthe LAC4 promoter as well as the chymosin expression-cassette, inbetween the LAC4 promoter and terminator sequences as present inpGBamdS6 (FIG. 40). Plasmid pKS105 contains the prochymosin cDNA fusedto the prepro-region of S.cerevisiae α-factor under control of the LAC4promoter (van den Berg et al. (1990) Bio/Technology 8, 135-139). Using aPCR, additional restriction sites were introduced at the 5' and 3' endof the fusion LAC4 promoter and chymosin expression-cassette. In the PCRpKS105 DNA was used as template and oligonucleotides AB3965 (SEQ ID NO:34) and AB3966 (SEQ ID NO: 35) were used as primers:

Oligo AB3965 (SEQ ID NO: 34)

5'-CTGCTACGTAATGTTTTCATTGCTGTTTTAC-3' (a LAC4 promoter-specific sequencestarting at the restriction site SnaB1)

Oligo AB3966 (SEQ ID NO: 35)

5'-CCGCCCAGTCTCGAGTCAGATGGCTTTGGCCAGCCCC-3' (chymosin-specific sequencewith the additional restriction site Xho1).

The PCR was performed using standard conditions and the obtained PCRfragment was digested with SnaB1 and Xho1. The plasmid pGBamdS6 waspartially digested with XhoI and subsequently digested with SnaBI andthe 10.9 kb DNA fragment was isolated and purified bygel-electrophoresis. The SnaB1-Xho1 fusion fragment LAC4promoter/chymosin expression-cassette was cloned into the SnaB1/Xho1sites of pGBamdS6. The resulting plasmid was designated pGBamdS7 (FIG.40).

To destroy the HindIII site approximately 66 bp upstream the startcodonfrom the chymosin gene, pGBamdS7 was partially digested with HindIII andtreated with the Klenow fragment of E.coli DNA polymerase I to generateblunt ends, subsequently ligated and transferred to E.coli for molecularcloning. The derived plasmid was designated pGBamdS8 and contains a LAC4promoter fragment with a destroyed HindIII site.

EXAMPLE 9 Expression of the amdS cDNA from the LAC4 Promoter in theYeast K.lactis

The expression vector, pGBamdS1, contains, apart from the amdS cDNA asecond selection marker which confers resistance to the antibiotic G418.This allows to first select for transformants using G418 resistancewhich is a well established procedure (Sreekrishna et al. (1984) Gene28, 73-81). The transformants obtained this way can subsequently be usedto verify expression of the amdS cDNA and to optimize conditions forselection of the amdS⁺ phenotype in K.lactis. Once these conditions havebeen established, direct selection for amdS⁺ transformants can beperformed, e.g. using expression cassettes without additional selectionmarkers.

pGBamdS1 (FIG. 34) was linearized in the LAC4 promoter by SstIIdigestion. The pTZ19R sequences were removed by fractionation in andpurification from agarose gels. 15 μg's of this DNA fragment were usedto transform to the K.lactis strains CBS 2360 and CBS 683 as describedby Ito H. et al. (1983) J. Bacteriol. 153, 163-168 with themodifications described under Experimental. The transformation plateswere incubated at 30° C. for 3 days. G418-resistant transformants wereobtained with both strains. Several independent transformants of bothstrains as well as the wild type strains were subsequently streaked ontoplates containing different solid media (see Table 1). YEPD andYEPD/G418 have been described in Experimental. YCB, YCB/NH₄ andYCB/acetamide have been described in example 7 as media I, II and III,respectively. YNB-lac/NH₄ and YNB-lac/acetamide contain 0.17% (w/v)Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco)supplemented with 1% (w/v) lactose, 30 mM Sodium Phosphate buffer at pH7.0 and either 0.1% (w/v) NH₄ (SO₄)₂ or 5 mM acetamide, respectively.

The amdS⁺ phenotype of the CBS 683/pGBamdS1 transformants was obvious onYCB/acetamide (see Table 1). However, the CBS 2360 transformantscontaining the same expression vector did not show any growth onYCB/acetamide. We reasoned that this might be due to the lack ofinduction of the LAC4 promoter driving the amdS cDNA in the absence oflactose or galactose as carbon-source dependent differences in theregulation of the LAC4 promoter between different K.lactis strains havebeen described (Breunig (1989) Mol. Gen. Genet. 216, 422-427). Table 1shows that this is indeed the case, on medium containing lactose as solecarbon-source and acetamide as sole nitrogen source the CBS 2360transformants were able to grow. We can therefore conclude that,depending on the carbon-source used, these transformants sufficientlyexpress the A.nidulans amdS cDNA in order to sustain growth of the yeastK.lactis on acetamide as sole nitrogen-source.

Southern analyses were performed to verify whether integration in theLAC4 promoter had occurred. High molecular weight DNA of several CBS2360 and CBS 683 transformants was isolated, digested with HindIII andsubsequently fractionated by electrophoresis on a 0.7% agarose gel.After transfer to nitrocellulose, hybridization was performed accordingto standard procedures. As probe a ³² P-labelled approximately 1.5 kbSacII/HindIII LAC4 promoter fragment isolated from plasmid pGBHSA20(FIG. 34) was used. We identified CBS 683 and CBS 2360 transformantscontaining a single pGBamdS1 expression cassette integrated in the LAC4locus, one example of each is shown in FIG. 41 and is designated KAM-1and KAM-2, respectively. Single copy integration of pGBamdS1 in the LAC4promoter produces two new HindIII fragments of 4.2 and 8.6 kb, both ofwhich are present in transformants KAM-1 and KAM-2. Since CBS 683contains two LAC4 loci and pGBamdS1 has integrated in only one of themin KAM-1, the digest of KAM-1 also shows the 5.6 kb HindIII fragmentderived from the second undisturbed LAC4 locus.

                  TABLE 1    ______________________________________    Growth of K. lactis CBS 683 and CBS 2360 wild type and    pGBamdS1 transformants on solid media containing different    nitrogen- and/or carbon-sources.    strain     CBS 683        CBS 2360    transforming DNA               none    pGBamdS1   none  pGBamdS1    ______________________________________    YEPD       +       +          +     +    YEPD-G418  -       +          -     +    YCB        -       -          -     -    YCB/NH.sub.4               +       +          +     +    YCB/acetamide               -       +          -     -    YNB-lac/NH.sub.4               +       +          +     +    YNB-lac/acetamide               -       +          -     +    ______________________________________

EXAMPLE 10 Direct Selection of K.lactis CBS 683 and CBS 2360Transformants Using Acetamide as Sole Nitrogen-source

SstII linearized pGBamdS1 (15 μg) was transformed into K.lactis CBS 683and CBS 2360 using the transformation procedure as described by Ito H.et al. ((1983). J. Bact. 153, 163-168.) with the followingmodifications:

K.lactis cultures were harvested for transformation at OD₆₁₀ =0.5-1.0.

After the 5 minutes heatshock of the DNA-cell suspension, the phenotypicexpression prior to plating was performed for 150-180 minutes at 30° C.in volumes of 1 ml. Different media were used for both strains. For CBS683 a YEPD/YNB solution (1*YNB (Yeast Nitrogen Base, Difco), 1%bacto-peptone, 1% yeast extract and 2% glucose) or YNB-glu (1*YNB (YeastNitrogen Base w/o Amino Acids and Ammonium Sulphate, Difco) supplementedwith 1% (w/v) glucose and 30 mM Sodium Phosphate buffer at pH 7.0) wereused. After this incubation the cells were centrifuged at 2000 g at roomtemperature for 5 minutes and subsequently plated on YCB/acetamide (seeexample 7). For CBS 2360, YNB-lac (1*YNB (Yeast Nitrogen Base w/o AminoAcids and Ammonium Sulphate, Difco) supplemented with 1% (w/v) lactoseand 30 mM Sodium Phosphate buffer at pH 7.0) was used. After thisincubation the cells were centrifuged at 2000 g at room temperature for5 minutes and subsequently plated on YNB-lac/acetamide (see example 9).

Growth was performed at 30° C. for 3 days. amdS⁺ transformants wereobtained for both strains. The transformation frequencies found werecomparable to that found when using the G418 selection. The correctidentity of the transformants was confirmed by subsequent plating onYEPD-plates containing G418 and by Southern analysis.

pGBamdS3 (FIG. 36), in which the amdS cDNA is driven from the KlEF1promoter, was linearized in the LAC4 terminator by digestion with XhoIand 15 μg of the gel-isolated fragment was subsequently transformed intothe K.lactis strain CBS 683 using direct selection on YCB/acetamideplates as described above for the transformation of pGBamdS1 into CBS683. Some of the transformants obtained were analyzed by Southernblotting. High molecular weight DNA was isolated, digested with BamHIand subsequently separated by electrophoresis on a 0.7% agarose gel.Following transfer to nitrocellulose, hybridization was performedaccording to standard procedures. As probe the ³² P-labelled 1.2 kbSphI/HindIII LAC4 terminator fragment isolated from plasmid pTY75LAC4(described in Example 8) was used. The results of several CBS 683transformants, containing the pGBamdS3 plasmid and from severaltransformants containing the pGBamdS5 plasmid are shown in FIGS. 42A and42B respectively. The reference strain CBS 683 is shown in FIG. 42B. Inthe CBS 683 transformants an additional 6.8 kb sized hybridizingfragment is present besides the 3.7 kb hybridizing fragment of theintact LAC4 terminator. This implicates a correct integration of theplasmids into the LAC4 terminator region.

In all of these transformants pGBamdS3 was integrated in one or morecopies into the LAC4 terminator (the intensity of the 6.8 kb hybridizingfragment is an indication for the number of integrated copies of thevector). We conclude that also the constitutive KlEF1 promoter can drivethe amdS cDNA for use as selection marker. Similar results were obtainedwith pGBamdS5 (FIG. 37), in which the amdS cDNA is driven from theS.cerevisiae ADH1 promoter.

EXAMPLE 11 Transformation of S.cerevisiae With pGBamdS5 by DirectSelection on Acetamide

In this example we have tested whether the amdS cDNA can also be used asselection marker in other yeasts, e.g. S.cerevisiae. We have firstestablished the amdS⁻ phenotype of S.cerevisiae strain D237-10B and itsability to use ammonium as sole nitrogen-source, using the same mediaand procedures as we have described for K.lactis in example 7. Asobserved in the case of K.lactis, full grown S.cerevisiae colonies wereonly observed on the plates containing ammonium as nitrogen source. Inplates without nitrogen-source or with acetamide as sole nitrogen-sourceno growth or, occasionally a slight background growth was observed.Plasmid pGBamdS5 was linearized in the ADH1 promoter by partialdigestion with SphI. The S.cerevisiae strain D273-10B (ATCC 25657) wastransformed with 15 μg of gel-isolated linearized pGBamdS5 fragment,using transformation procedures as described in example 10 for thetransformation of pGBamdS1 to K.lactis CBS 683. After transformation thecells were plated onto YCB/acetamide plates (see Example 9) and allowedto grow at 30° C. for 3 days. Several amdS⁺ transformants were obtainedwith in this transformation. Subsequent Southern analysis of some of theamdS transformants confirmed that the amdS cDNA was stably integratedinto the S.cerevisiae genome.

High molecular weight DNA was isolated and digested with BamHI,subsequently separated by electrophoresis on a 0.7% agarose gel andblotted onto nitrocellulose. As probe the ³² P labelled 750 bp EcoRVamdS fragment was used isolated from pGBamdS1. The results of severalD273-10B/pGBamdS5 transformants as well as the reference strain D273-10B(ATCC 25657) are shown in FIG. 43. Two hybridizing fragments are presentin the D273-10B transformants respectively, a 6.6 kb fragment thatrepresents the multicopy fragment and a hybridizing fragment of unknownsize that represents the flanking. The reference strain D273-10B (ATCC25657) as expected does not show any hybridizing fragment.

EXAMPLE 12 Removal of the amdS-marker From K.lactis and S.cerevisiaeamdS⁺ Transformants Using Fluoracetamide Counter-selection

In the above described examples the amdS containing expression cassettesare integrated by a single cross-over homologous recombination in theK.lactis and S.cerevisiae genomes. This means that the amdS cDNA isflanked by direct repeats in the genomes of these amdS yeasttransformants. Consequently, the amdS cDNA will be deleted in a smallfraction of the transformant population by intra-chromosomal mitoticrecombination events occurring at low frequency between the directrepeats flanking the cDNA. It should be possible to select for theseevents using media containing fluoracetamide, a compound which is toxicfor amdS⁺ cells but not for amdS⁻ cells as has been shown for A.nidulansby Hynes and Pateman ((1970) Mol. Gen. Genet. 108, 107-116). In amdS⁺cells fluoracetamide is converted into ammonium and fluoracetate, thelatter being toxic when activated by the enzyme acetyl-CoA-synthetase.Prerequisites for the fluoracetamide counter-selection to also work onamdS⁺ yeasts are therefore 1) fluoracetamide should not be toxic foramdS⁻ yeasts, 2) the yeast cellwall and plasmamembrane should bepermeable to fluoracetamide and 3) the enzyme acetyl-CoA-synthetaseshould be active. To test this we have used a K.lactis CBS 683transformant containing a single copy of pGBamdS1 integrated in the LAC4promoter, designated KAM-1 and a S.cerevisiae D273-10B transformantcontaining a single copy of pGBamdS5 integrated in the ADH1 promoter,designated SAM-1. Both KAM-1 and SAM-1 were subjected to at least 3rounds of genetic purification on selective medium (YCB/acetamide) toexclude contamination with the amdS⁻ parental strains. KAM-1 and SAM-1were each plated at a density of approximately 10³ CFU per plate ontoYCB/NH₄ supplemented with 10 mM fluoracetamide. For both KAM-1 andSAM-1, 5 to 20 fluoracetamide resistant colonies appeared after 3 to 6days at 30° C. Southern analysis on chromosomal DNA from severalindependent KAM-1 and SAM-1 derived amdS⁻ colonies confirmed that theamdS cDNA was correctly removed from the K.lactis and S.cerevisiaegenomes by homologous recombination between the flanking direct repeats(FIG. 41). In fact, in one of the KAM-1 amdS⁻ recombinants thecrossover-point of the recombination was located between a polymorphicHindIII site and the amdS cDNA. This polymorphic HindIII is present 92bp upstream of the LAC4 reading frame in the LAC4 promoter of pGBamdS1,however, this site is not present in the CBS 683 LAC4 promoter. Therecombination event has left the HindIII site in the genome of thisparticular KAM-1 recombinant which could otherwise not be discriminatedfrom the parent strain CBS 683 (see the extra 4.2 kb fragment in FIG.41, lane 6). This KAM-1 recombinant therefore excludes the possibilitythe we would have isolated CBS 683 contaminants in stead of KAM-1 amdS⁻recombinants. We conclude from the above that the amdS cDNA can beremoved from yeast genomes when flanked by direct repeats usingfluoracetamide counter-selection. In the present example the amdS⁻K.lactis and S.cerevisiae recombinants occur at a frequency of about0.1%.

We have noted that for some yeast strains efficient counter-selection onfluoracetamide cannot be performed on YCB/NH₄, probably due to strongcarbon-catabolite repression of the acetyl-CoA-synthetase. In thoseinstances we have successfully used YNB-galactose/NH₄ (this medium isidentical to YNB-lac/NH₄ described in example 9 but contains 1%galactose in stead of 1% lactose) supplemented with 10 mM fluoracetamidefor counter-selection.

EXAMPLE 13 Marker Gene Free Deletion of a K.lactis Gene Using theamdS-marker

A frequently used technique for the manipulation of yeast genomes is"one-step gene disruption", a method which allows to disrupt (or modify)genes in a single transformation step (Rothstein et al. (1983) MethodsEnzymol. 101, 202-211). In this method a transforming plasmid with acopy of a target gene disrupted by a yeast selectable marker integratesinto the yeast genome via a double cross-over homologous recombination,resulting in the replacement of the wild-type target gene by thedisrupted copy. Combination of "one-step gene disruption" and thefluoracetamide counter-selection of amdS⁺ -yeast-transformants as wehave described in Example 12, should enable the deletion of genes fromyeast genomes without leaving selectable markers. In this example wehave used this combination to delete the LAC4 gene from the K.lactis CBS2360 genome. For one-step gene transplacement of the K.lactis LAC4 genepGBamdS6 (FIG. 38) was constructed, which contains the amdSexpression-cassette flanked by LAC4 promoter and terminator sequences.An additional LAC4 terminator fragment is present directly upstream ofthe amdS expression-cassette such that the amdS expression-cassette isflanked by direct repeats which will allow the excision of the amdSsequences from the K.lactis genome by intrachromosomal recombinationbetween these direct repeats. Plasmid pGBamdS6 was digested with SpeIand HindIII and a 6.6 kb DNA fragment was isolated after gelelectrophoresis. This SpeI-HindIII fragment, containing the genereplacement vector, was used to transform K.lactis CBS 2360 usingtransformation procedures described in Example 10. amdS⁺ transformantswere plated onto on YEPD plates containing 0.008% X-gal(5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) in order to screenfor transformants with a transplaced LAC4 gene.

The amdS+ transformants were analyzed on Southern blot. High molecularweight DNA was isolated, digested with HindIII, subsequently separatedby electrophoresis on a 0.7% agarose gel and blotted ontonitrocellulose. As probe a ³² -P-labelled 600 bp XbaI LAC4 terminatorfragment isolated from plasmid pPTLAC4 (described in example 8) wasused. The results of an amdS+ CBS 2360 transformant with a transplacedLAC4 gene as well as the reference strain CBS 2360 are shown in FIG. 44.In case of the amdS+ CBS 2360 transformant, a 7.4 kb hybridizingfragment is present that implicates a correctly transplaced LAC4 gene.The reference strain CBS 2360 shows a 2.0 kb hybridizing fragment thatrepresents the intact LAC4 locus.

Subsequent fluoracetamide counter-selection of these amdS⁺ transformantsas described in Example 12, yielded recombinants with an amdS⁻phenotype. Southern analysis was performed on the chromosomal DNA of theamdS⁻ recombinants. High molecular weight DNA was isolated, digestedwith HindIII, subsequently separated on a 0.7% agarose gel and blottedonto nitrocellulose. The same ³² P-labelled probed as described abovewas used. The results of the amdS- CBS 2360 recombinants are shown inFIG. 44. In case of the amdS- recombinants, a 5.4 kb hybridizingfragment is present, which confirmed the absence of the LAC4 gene aswell as the correct removal of the amdS marker from the yeast genome.The absence of the amdS marker from these K.lactis LAC4⁻ strains offersthe possibility to reuse the amdS marker for additional deletions and/ormodifications of genes.

EXAMPLE 14 Marker gene free insertion of a gene into the K.lactis genomeusing the amdS marker

For the marker gene free insertion of genes into the yeast genome wehave used the chymosin cDNA as a model-gene. In this example we haveinserted the chymosin CDNA at the K.lactis LAC4 locus while replacingthe LAC4 gene and without leaving a selection marker. The principle ofmarker-free gene insertion is the same as that for marker-free deletionof genes as described in example 13 except that in this case thetransplacement vector pGBamdS8 contains a gene of interest, the chymosincDNA (FIG. 40). Plasmid pGbamdS8 was digested with SpeI and HindIII andthe 8.0 kb DNA fragment was gel-isolated. 10 μg of this fragment wastransformed to K.lactis CBS 2360 as described in Example 10. amdS⁺transformants with a transplaced LAC4 gene and chymosin activity wereobtained. Chymosin activity was measured as described (van den Berg etal. (1990) Bio/technology 8, 135-139). By subsequent counter-selectionof these transformants on fluoracetamide as described in example 12recombinants were isolated with an amdS⁻ phenotype but which stillproduced chymosin. Southern analysis of the chromosomal DNA of theamdS⁻, Chymosin⁺ recombinants confirmed the replacement of the LAC4 geneby the chymosin cDNA as well as the correct removal of the amdS markerfrom the K.lactis genome. The amdS⁻ /chymosin⁺ phenotype of theserecombinants was also confirmed by lack of growth on YCB/acetamideplates and by the presence of chymosin activity (see above). The amdS⁻phenotype of these recombinants allows further manipulation of thesestrains using the amdS marker, e.g. integration of additional copies ofthe chymosin expression-cassette and/or deletion of K.lactis genes asdescribed in example 13.

EXAMPLE 15 Test of the amdS-minus phenotype of Bacilli and E.coli

A prerequisite for the use of the amdS selection system in Bacilli isthat these Gram-positive bacteria do not contain any acetamidaseactivity. In order to test this we have plated the B.subtilis strainBS-154 (CBS 363.94) on a minimal Bacillus medium containing all theessential nutritives and vitamins except a nitrogen-source (28.7 mM K₂HPO₄, 22 mM KH₂ PO₄, 1.7 mM sodium citrate, 0.4 mM MgSO₄, 0.75 μM MnSO₄,0.5% (w/v) glucose and 1.5% agar. No growth was observed on this mediumas such or when supplemented with 20 mM acetamide as nitrogen-source.Growth was only observed in the case that the minimal medium wassupplemented with either 20 mM (NH₄)₂ SO₄ or 20 mM KNO₃ as nitrogensource. We conclude that Bacillus BS-154 (CBS 363.94) lacks sufficientacetamidase activity to sustain growth on acetamide as sole nitrogensource. This phenomenon should allow for the A.nidulans amdS gene to beused as selection marker in Gram-positive bacteria.

Similarly we have tested the lack of acetamidase activity in aGram-negative bacterium, in this case E.coli, in order to establishwhether the A.nidulans amdS gene can also be used as selection marker inthese micro-organisms. In this case we used M9 minimal medium (Sambrooket al. (1989) "Molecular Cloning: a laboratory manual", Cold SpringHarbour Laboratories, Cold Spring Harbour, N.Y.) supplemented with 0.02μg (w/v) thiamine. Full grown colonies of E.coli JM109 were observed onwhen plated on M9 plates. No growth or only slight background growth wasobserved, however, when the NH₄ Cl was omitted from the M9 plates orreplaced by 20 mM acetamide. We conclude that the E.coli JM109 strainlacks sufficient acetamidase activity to sustain growth on acetamide assole nitrogen source. This should allow for the A.nidulans amdS gene tobe also used as selection marker in Gram-negative bacteria.

EXAMPLE 16 Construction of amdS expression-vectors for use in bacteria

Construction of pGBamdS22

To express the A.nidulans amdS gene in different Bacilli species, wehave cloned the amdS cDNA from pamdS-1 into the basic Bacillusexpression vector pBHA-1 (European Patent Application 89201173.5; FIG.45 for physical map). At the ATG initiation-codon of the amdS cDNA genean NdeI site was introduced in pamdS-1 using oligonucleotides AB3825(SEQ ID NO: 36) and AB3826 (SEQ ID NO: 37) with the following sequences:

Oligo AB3825 (SEQ ID NO: 36)

5'-CGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATATGT-3' Oligo AB3826 (SEQ IDNO: 37):

5'-CTAGACATATGCCTCAATCCTGGGAAGAACTGGCCGCTGATAAG-3'

Annealing of these oligonucleotides was carried out using standardprocedures. The resulting double stranded DNA fragment was ligated intoBssHII/XbaI digested pamdS-1 and transferred to E.coli. From one of thetransformants pGBamdS21 was isolated and characterized byrestriction-enzyme analysis (FIG. 47). pGBamdS21 was digested with KpnIand HindIII and the amdS cDNA containing fragment was cloned into pBHA-1digested with KpnI and HindIII. The resulting plasmid was designatedpGBamdS22 (FIG. 48).

Construction of pGBamdS25

To demonstrate site specific integration of a desired DNA sequence intothe B.licheniformis genome using amdS as selection marker, the amdS cDNAwas cloned in the expression/integration-vector pLNF (FIG. 46). Thisvector containing the 5' and 3' non-coding sequences of theB.licheniformis amylase gene enables site specific integration at thecorresponding chromosomal amylase locus. pGBamdS21 (described above,FIG. 47) was digested with NdeI and PvuII and the amdS cDNA containingfragment was ligated with pLNF digested with NdeI and ScaI. The ligationmixture was transformed to B.subtilis BS-154 (CBS 363.94). Transformantswere selected on minimal medium supplemented with 20 μg/ml neomycin.From one of the transformants, designated BAA-101, the plasmid pGBamdS25(FIG. 50) was isolated.

Construction of pGBamdS41

For the expression of the A.nidulans amdS CDNA in E.coli we have usedpTZ18R/N, a derivative of pTZ18R which is described in the EuropeanPatent Application 0 340 878 A1 pTZ18R/N differs from pTZ18R in that anNdeI site was created at the ATG start-codon of the lacZ reading framein pTZ18R using in vitro site directed mutagenesis. pGBamdS21 wasdigested with NdeI and HindIII and the gel-isolated fragment containingthe amdS CDNA was ligated into pTZ18R/N digested with NdeI and HindIII.This ligation mixture was used to transform E.coli JM109 and from one ofthe transformants pGBamdS41 (FIG. 51) was isolated.

EXAMPLE 17 Transformation of Bacilli using the amdS gene as selectionmarker

In order to delete the E.coli sequences from pGBamdS22 and to place the"hpa2"-promoter immediately upstream of the amdS cDNA, pGBamdS22 wasdigested with NdeI, recircularized by ligation and used to transformB.subtilis BS-154 (CBS 363.94). Transformants were selected on acetamideminimal plates and checked for neomycin resistance. From one of thesetransformants expression-vector pGBamdS23 (FIG. 49) was isolated andcharacterized by restriction enzyme analysis. These results show that 1)the A.nidulans amdS cDNA under the control of a Bacillus promotersequence is expressed well and 2) that the amdS gene can be used as aselection marker in the transformation of Bacilli.

B.licheniformis T5 (CBS 470.83) was transformed with vector pGBamdS25.Transformation was performed as described in Experimental and amdS⁺transformants were obtained by direct selection on modified protoplastregeneration plates supplemented with 20 mM acetamide as sole nitrogensource (described in Experimental). The presence of pGBamdS25 in thetransformants was confirmed by their neomycin resistance phenotype aswell as the fact that the plasmid could be reisolated from thetransformants. One of these transformants designated BAA-103 was used toachieve integration of plasmid pGBamdS25 into the B.licheniformis genometargeted at the amylase locus. Plasmid integration was performed bygrowing transformants at 50° C. on minimal medium agar containingacetamide as sole nitrogen source. Several colonies were transferredrepeatedly (2 to 3 times) to fresh plates followed by incubation at 50°C. Isolated colonies were tested for their ability to grow on acetamideas sole nitrogen source and for resistance to neomycin at 1 μg/ml. Theabsence of autonomously replicating plasmid DNA was established byre-transformation of DNA isolated from the integrants to the hoststrain. No neomycin resistant colonies could be obtained.

This result is a clear evidence that the amdS gene is a suitable markerto select Bacillus species containing a single amdS gene copy.

EXAMPLE 18 Transformation of E.coli using the amdS gene as selectionmarker

E.coli JM109 was transformed with the vector pGBamdS41 using standardprocedures. Selections were performed on either M9 plates supplementedwith 0.02 μg/ml thiamine and 50μg/ml ampicillin or M9 plates withoutammonium but supplemented with 20 mM acetamide, 0.02 μg/ml thiamine and0.05 mM IPTG. Several amdS⁺ /ampicillin resistant transformants wereobtained from which pGBamdS41 could be reisolated. The transformationfrequencies using selection on ampicillin or acetamide were comparable.This demonstrates that the A.nidulans amdS gene is functional asselection marker for the transformation of Gram-negative bacteria aswell.

EXAMPLE 19 Fluoracetamide counter-selection of amdS⁺ bacterialtransformants

Counter-selection of bacterial amdS⁺ transformants using fluoracetamiderequires the activity of the enzyme acetylCoA synthetase for theconversion of fluoracetate to fluoracetyl-CoA. To avoid cataboliterepression of acetylCoA synthase, as has been observed in E.coli (Brownet al., 1977), bacterial amdS⁺ transformants or single copy integrantswere grown on defined media containing NH₄ Cl as nitrogen source andacetate as carbon and energy source.

Many organisms including B. subtilis (Freese, E. and Fortnagel, P.(1969) J. Bacteriol 90, 745-756) lack a functional glyoxylate shunt andtherefore metabolize acetate only when the medium is supplemented with asource of TCA cycle intermediates, such as glutamate or succinate.Bacillus amdS⁺ strains were grown on TSS medium with 0.01% glutamate and50 mM acetate as described by Grundy, F. J. et.al. (1993) MolecularMicrobiology 10, 259-271. To this medium solidified with agar,fluoracetamide was added in concentrations ranging from 1 to 50 mM.B.subtilis BAA-101 or B.licheniformis BAA-103 (single copy integrant)were plated at a density of 10² cells per plate. At a certainfluoracetamide concentration only a few colonies appeared. The absenceof pGBamdS25 in these colonies was demonstrated by plasmid andchromosomal DNA analysis, sensitivity towards neomycin, and inability togrow on acetamide as sole nitrogen-source. Counter-selection of BAA-103in some cases led to the loss of the amylase gene as indicated byactivity assays and Southern blots. This shows that fluoracetamidecounter-selection can be used to select amdS⁻ cells from a populationcontaining a majority of amdS⁺ Bacillus cells and the simultaneousdeletion of a specific target gene.

Similarly we have used minimal medium #132 as described by VanderwinkelE. and De Vlieghere M, European J. Biochem, 5 (1968) 81-90 supplementedwith fluoracetamide in concentrations ranging from 1 to 50 mM and 0.05mM IPTG to select amdS⁻ E.coli JM109 cells from a population ofpGBamdS41 transformants. Cells were plated at a density of 10² cells perplate. At a certain fluoracetamide concentration only a few coloniesappeared. The absence of pGBamdS41 from the fluoracetamide selectedcolonies was confirmed by isolation of DNA, sensitivity towardampicillin and inability to grow on acetamide as sole nitrogen-source.This demonstrates that the fluoracetamide counter-selection can be usedto select amdS⁻ cells from a population containing a majority of amdS⁺E.coli cells.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 37    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3100    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CTAATCTAGAATGCCTCAATCCTGAA26    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3101    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GACAGTCGACAGCTATGGAGTCACCACA28    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: TN0001    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TCGATTAACTAGTTAA16    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 35 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB2154    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    AACCATAGGGTCGACTAGACAATCAATCCATTTCG35    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 35 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB2155    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GCTATTCGAAAGCTTATTCATCCGGAGATCCTGAT35    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB2977    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    TATCAGGAATTCGAGCTCTGTACAGTGACC30    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB2992    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GCTTGAGCAGACATCACCATGCCTCAATCCTGGGAA36    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB2993    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    TTCCCAGGATTGAGGCATGGTGATGTCTGCTCAAGC36    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB2994    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CTGATAGAATTCAGATCTGCAGCGGAGGCCTCTGTG36    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3657    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    AGCTTGACGTCTACGTATTAATGCGGCCGCT31    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3658    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    TCGAAGCGGCCGCATTAATACGTAGACGTCA31    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3779    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    AATTGGGGCCCATTAACTCGAGC23    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3780    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    AATTGCTCGAGTTAATGGGCCC22    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3448    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GTGCGAGGTACCACAATCAATCCATTTCGC30    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3449    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    ATGGTTCAAGAACTCGGTAGCCTTTTCCTTGATTCT36    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3450    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    AGAATCAAGGAAAAGGCTACCGAGTTCTTGAACCAT36    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3520    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    ATCAATCAGAAGCTTTCTCTCGAGACGGGCATCGGAGTCCCG42    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3781    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    AATTGGGGCCCAGCGTCC18    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3782    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    AATTGGACGCTGGGCCCC18    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3746    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    TGACCAATAAAGCTTCTCGAGTAGCAAGAAGACCCAGTCAATC43    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3747    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    CTACAAACGGCCACGCTGGAGATCCGCCGGCGTTCGAAATAACCAGT47    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB4234    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GAAGACCCAGTCAAGCTTGCATGAGC26    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB4235    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    TGACCAATTAAGCTTGCGGCCGCTCGAGGTCGCACCGGCAAAC43    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 69 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB4236    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    TGACCAATAAAGCTTAGATCTGGGGGTGATTGGGCGGAGTGTTTTGCTTAGACAATCAAT60    CCATTTCGC69    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB4233    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    TGACCAATAGATCTAAGCTTGACTGGGTCTTCTTGC36    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3514    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    CTGCGAATTCGTCGACATGCCTCAATCCTGGG32    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3515    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    GGCAGTCTAGAGTCGACCTATGGAGTCACCACATTTC37    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3701    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    CTGCGAATTCGTCGACACTAGTGGTACCATTATAGCCATAGGACAGCAAG50    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 70 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3700    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    GCTCTAGAGCGCGCTTATCAGCTTCCAGTTCTTCCCAGGATTGAGGCATTTTTAATGTTA60    CTTCTCTTGC70    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3702    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    CTGCGAATTCGTCGACACTAGTGGTACCATCCTTTTGTTGTTTCCGGGTG50    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 70 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3704    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    GCTCTAGAGCGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATTGTATATGAGA60    TAGTTGATTG70    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 55 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3704    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    GCTCTAGAAGTCGACACTAGTCTGCTACGTACTCCAGAATTTATACTTAGATAAG55    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3705    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    TGCTCTAGATCTCAAGCCACAATTC25    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3965    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    CTGCTACGTAATGTTTTCATTGCTGTTTTAC31    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3966    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    CCGCCCAGTCTCGAGTCAGATGGCTTTGGCCAGCCCC37    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3825    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    CGCGCTTATCAGCGGCCAGTTCTTCCCAGGATTGAGGCATATGT44    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (vii) IMMEDIATE SOURCE:    (B) CLONE: AB3826    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    CTAGACATATGCCTCAATCCTGGGAAGAACTGGCCGCTGATAAG44    __________________________________________________________________________

We claim:
 1. A method for obtaining a recombinant strain containing adesired DNA fragment and free of a selection marker, said methodcomprising the steps of:a) obtaining transformants of a bacterial,filamentous fungal or yeast host strain by introducing into said hoststrain a DNA molecule comprising a desired DNA fragment and anacetamidase gene that is the selection marker, wherein said acetamidasegene is dominant with respect to said host strain, b) selectingtransformants containing said desired DNA fragment and said acetamidasegene by selecting for the presence of said acetamidase gene, c)culturing said selected transformants under condition effecting deletionof said acetamidase gene, and d) selecting a recombinant straincontaining said desired DNA fragment in which said acetamidase gene hasbeen deleted.
 2. The method according to claim 1 wherein said desiredDNA fragment comprises a DNA molecule selected from the group consistingof a gene, a cDNA molecule, a promoter sequence, a translationterminator sequence, a regulatory element sequence, an intron sequence,a recognition sequence for a DNA-binding protein, atranslation-initiation site and a restriction site.
 3. The methodaccording to claim 1 wherein steps a) to d) are repeated on therecombinant strain obtained in step d), using either the same or adifferent desired DNA fragment.
 4. The method according to claim 1wherein the acetamidase gene is that of a fungal species.
 5. The methodaccording to claim 4 wherein said fungal species is an Aspergillusspecies.
 6. The method of claim 1, wherein said host strain is a fungalspecies.
 7. The method of claim 6, wherein said host strain is afilamentous fungal species.
 8. The method of claim 7, where said hoststrain is an Aspergillus species.
 9. The method according to claim 6,wherein the acetamidase gene is that of a fungal species.
 10. The methodaccording to claim 9, wherein the acetamidase gene is that of anAspergillus species.
 11. The method according to claim 7, wherein theacetamidase gene is that of a fungal species.
 12. The method accordingto claim 11, wherein the acetamidase gene is that of an Aspergillusspecies.
 13. The method according to claim 8, wherein the acetamidasegene is that of a fungal species.
 14. The method according to claim 13,wherein the acetamidase gene is that of an Aspergillus species.
 15. Themethod according to claim 6, wherein said desired DNA fragment comprisesa DNA molecule selected from the group consisting of a gene, a cDNAmolecule, a promoter sequence, a translation terminator sequence, aregulatory element sequence, an intron sequence, a recognition sequencefor a DNA-binding protein, a translation-initiation site and arestriction site.
 16. The method according to claim 7, wherein saiddesired DNA fragment comprises a DNA molecule selected from the groupconsisting of a gene, a cDNA molecule, a promoter sequence, atranslation terminator sequence, a regulatory element sequence, anintron sequence, a recognition sequence for DNA-binding protein, atranslation-initiation site and a restriction site.
 17. The methodaccording to claim 8, wherein said desired DNA fragment comprises a DNAmolecule selected from the group consisting of a gene, a cDNA molecule,a promoter sequence, a translation terminator sequence, a regulatoryelement sequence, an intron sequence, a recognition sequence for aDNA-binding protein, a translation-initiation site and a restrictionsite.
 18. The method according to claim 6, wherein steps a) to d) arerepeated on the recombinant strain obtained in step d), using either thesame or a different desired DNA fragment.
 19. The method according toclaim 7, wherein steps a) to d) are repeated on the recombinant strainobtained in step d), using either the same or a different desired DNAfragment.
 20. The method according to claim 8, wherein steps a) to d)are repeated on the recombinant strain obtained in step d), using eitherthe same or a different desired DNA fragment.
 21. The method of claim 1,wherein said selection marker gene is flanked by nucleotide sequencerepeats, and wherein deletion of the selection marker gene is effectedby recombination between said repeats.
 22. The method according to claim21 wherein said flanking nucleotide sequence repeats consist of anucleotide sequence contained in the DNA of said host strain.
 23. Themethod of claim 21, wherein said host strain is a fungal species. 24.The method according to claim 23, wherein said flanking nucleotidesequence repeats consist of a nucleotide sequence contained in the DNAof said host strain.
 25. The method of claim 23, wherein said hoststrain is a filamentous fungal species.
 26. The method according toclaim 25, wherein said flanking nucleotide sequence repeats consist of anucleotide sequence contained in the DNA of said host strain.
 27. Themethod of claim 25, where said host strain is an Aspergillus species.28. The method according to claim 27, wherein said flanking nucleotidesequence repeats consist of a nucleotide sequence contained in the DNAof said host strain.
 29. A method for introducing an alteration into atarget DNA sequence contained in a bacterial, filamentous fungal oryeast host strain which method comprises:(a) obtaining transformants ofsaid host strain by introducing into said host strain a vectorcomprising an acetamidase selection marker gene, flanked by nucleotidesequence repeats to form a marker cassette, wherein said repeats consistof a nucleotide sequence immediately 5' or a nucleotide sequenceimmediately 3' of said target DNA sequence; and further wherein whensaid flanking nucleotide sequence repeats consist of a nucleotidesequence 5' of said target DNA sequence, said vector further comprises,3' of, and contiguous to, said marker cassette, an additional nucleotidesequence consisting of a nucleotide sequence 3' of said target DNAsequence; and wherein when said flanking nucleotide sequence repeatsconsist of a nucleotide sequence 3' of said target DNA sequence, saidvector further comprises, 5' of, and contiguous to, said markercassette, an additional nucleotide sequence consisting of a nucleotidesequence 5' of said target DNA sequence; (b) selecting transformants byselecting for the presence of said acetamidase marker gene; (c)culturing said selected transformants under condition effecting deletionof said acetamidase marker gene by recombination between said flankingnucleotide sequence repeats; and (d) selecting a recombinant strain withan alteration in said target DNA in which said acetamidase marker genehas been deleted from said recombinant strain.
 30. The method of claim29 wherein said acetamidase gene is that of a fungal species.
 31. Themethod of claim 30 wherein the fungal species is an Aspergillus species.